Arf and HDM2 interaction domains and methods of use thereof

ABSTRACT

The present invention discloses that the binding of Arf with Dm2 results in specific domains of both proteins undergoing a dramatic transition from disordered conformations to extended structures comprised of β-strands. The presence of these specific domains is necessary and sufficient for the formation of the highly stable extended β structures formed between these two proteins. The present invention further exploits this discovery by providing unique methods for identifying and/or designing compounds that mimic, inhibit and/or enhance the effect of Arf on Dm2. The present invention also provides specific protein fragments derived from Arf and Dm2 that play a critical role in the binding of these two important regulatory proteins.

RESEARCH SUPPORT

[0001] The research leading to the present invention was supported inpart by the American Cancer Society and a Cancer Center (CORE) SupportGrant CA21765. The government may have certain rights in the presentinvention. Support for this invention was also provided by the AMERICANLEBANESE SYRIAN ASSOCIATED CHARITIES and the ASSISI FOUNDATION OFMEMPHIS INC.

FIELD OF THE INVENTION

[0002] The present invention relates to the interaction of Arf and Hdm2,as well as to specific protein fragments derived from Arf and Hdm2 thatplay a critical role in the binding of these two regulatory proteins.The present invention also relates to the use of Arf and Hdm2 andspecific fragments thereof in unique assays for identifying compoundsthat can be used in the treatment of cancer. The present inventionfurther relates to identifying and using specific domains of a givenprotein that interact with one or more specific domains of a secondprotein.

BACKGROUND OF THE INVENTION

[0003] Disruption of cell cycle control mechanisms contributessignificantly to the development of cancer in humans [Sherr, CancerRes., 60:3689-95 (2000)]. Consistently, the two most frequentlyinactivated tumor suppressor genes in human cancer irrespective of tumortype, site, and patient age, are the p53 gene and the INK4a-Arf genelocus, both of which encode proteins involved in the regulation ofcellular replication [Hall and Peters, Adv. Cancer Res., 68:67-108(1996); Hainaut et al., Nucleic Acid Res., 25:151-157 (1997)]. The p53gene encodes the transcription factor p53. Activation of the p53 gene inresponse to oncogenic stress signals results in cell cycle arrest orapoptosis, thereby enabling cells to repair genotoxic damage oralternatively, to be eliminated from the organism [reviewed in Ko etal., Genes & Devel. 10:1054-1072 (1996); Levine, Cell 88:323-331(1997)]. Loss of p53 function cancels these surveillance functionsthereby allowing defective cells to replicate and predisposing the cellto cancer development.

[0004] The INK4a/Arf gene locus has been shown to encode two unrelatedproteins from alternative but partially overlapping reading frames: (i)p16^(Ink4a) and (ii) Arf (p14^(Arf) in humans and p19^(ARF) in themouse) [Quelle et al., Cell, 83:993-1000 (1995)]. These proteinsindependently target two cell cycle control pathways. The N-terminal 62amino acid residues of the 132 amino acid p14^(ARF) protein and theN-terminal 63 amino acid residues of the 169 amino acid p19^(ARF)protein are encoded by a unique first exon (1β), whereas the remainingamino acid residues are encoded by exon 2. An alternative reading frameof exon 2 also encodes the bulk of p16^(INK4a).

[0005] p16^(INK4a) is an antagonist of cell replication. Morespecifically, p16^(INK4a) inhibits the cyclin D-dependent kinases CDK4and CDK6 [Serrano et al., Nature, 366:704-707 (1993)]. CDK4 and CDK6play an important role in the cell replication cycle through theirphosphorylation of the retinoblastoma protein (Rb). Hyperphosphorylationof Rb stimulates the cell to exit from the G1 phase and begin DNAsynthesis, a required step prior to cell division. Thus, the inhibitionof CDK4 and CDK6 by p16^(INK4a) prevents hyperphosphorylatedRb-dependent DNA synthesis, thereby maintaining the cell in itsnon-replicating mode.

[0006] Disruption in mice of either the entire INVK4a/Arf locus [Serranoet al., Cell, 85: 27-37 (1996)] or exon 1β [Kamijo et al., Cell,91:649-59 (1997)] leads to multi-type tumor growth and early death,identifying Arf as a bona fide tumor suppressor. Interestingly, it hasbeen suggested that disruption of INK4a does not contribute tospontaneous tumor formation in mice and that Arf disruption accounts forthe high rate of spontaneous tumor formation in INK4a/Arf-null mice[Sherr, Cancer Res., 60: 3689-95 (2000)]. Since the INK4a/Arf locus isfrequently disrupted in human cancers [Raus and Peters, Biochim.Biophys. Acta Rev. Cancer, 1378:F115-F177 (1998)], the loss of Arffunction appears to be a major contributor to human cancers.

[0007] Indeed, Arf, in concert with other cell cycle regulators andtumor suppressors such as p53 and Rb, plays a central role in cellularresponses to oncogenic stress, such as inappropriate mitogenicsignaling. For example, Arf expression is activated by overexpression ofproteins involved in mitogenic signaling, such as Myc [Zindy et al.,Genes & Dev., 12:2424-2434 (1998)], E1A [de Stanchina et al., GenesDev., 12:2434-42 (1998)], E2F [Bates et al., Nature, 395:124-5 (1998)],Ras [Palmero et al., Nature, 395:125-6 (1998)], and v-Ab1 [Radfar etal., Proc. Natl. Acad. Sci. U.S.A., 95:13194-13199 (1998)]. Activationof Arf leads to stabilization of p53 [Pomerantz et al., Cell, 92:713-23(1998); Kamijo et al., Proc. Natl. Acad. Sci., 95:8292-8297 (1998);Stott et al., Embo J, 17: 5001-14 (1998); and Zhang et al., Cell,92:725-734 (1998)] followed by cell cycle arrest. Arf therefore connectsthe Rb and p53 pathways [Sherr, Cancer Res., 60: 3689-95 (2000)] so thatexcessive proliferative signaling via the Rb pathway activates arrestmechanisms controlled by p53.

[0008] Arf stabilizes p53 by interfering with an auto-regulatory loopinvolving p53 and Double Minute 2 (Hdm2 in humans, Mdm2 in mice) [Wu etal., Genes Dev., 7:1126-1132 (1993)] that maintains p53 at low levelsunder normal cellular conditions (i.e. in the absence of oncogenicstress, DNA damage, etc.). The positive component of thisauto-regulatory loop involves activation of Mdm2 transcription by p53[Barak et al., EMBO J, 12:461-468 (1993)]. The negative component hasseveral facets. First, Mdm2 binds p53 [Kussie et al., Science,274:948-953 (1996)] and inhibits the transactivation function of p53[Oliner et al., Nature, 362:857-860 (1993); Momand et al., Cell,69:1237-1245 (1992)]. Second, Mdm2 shuttles p53 from the nucleus to thecytoplasm and facilitates p53 degradation [Roth et al., Embo J,17:554-64 (1998)]; Freedman et al., Mol Cell Biol, 18:7288-93 (1998)].Third, Mdm2 acts as an E3 ubiquitin ligase toward p53 within theubiquitin-dependent 26S proteosome pathway [Honda et al., FEBSLett,420:25-7 (1997)]. Therefore, Mdm2 inhibits p53 activity in the nucleusthrough multiple and diverse mechanisms. Balance between the positiveand negative components of this auto-regulatory system is essential forcell survival. When p53 is inactivated, mice develop tumors at anunusually high rate [Donehower et al., Nature, 356:215-221 (1992)],indicating that p53-dependent tumor suppression is compromised.Additionally, when Mdm2 is inactivated, mice are not viable [Jones etal., Nature, 378:206-8 (1995); Montes de Oca Luna et al., Nature,378:203-206 (1995)], suggesting that unregulated p53 expression islethal. Mdm2^(−/−) mice are rescued, however, by the additionalinactivation of p53 [Jones et al., Nature, 378:206-8 (1995); Montes deOca Luna et al., Nature, 378:203-206 (1995)]. Thus, proper regulation ofp53 activity relies on appropriate balance between the positive andnegative components of the p53-Mdm2 auto-regulatory system.

[0009] The first direct biochemical connection between p19^(ARF) and p53was established when it was found that p19^(ARF) could bind to Mdm2,[Pomerantz et al., Cell, 92:713-723 (1998); Zhang et al., Cell,92:725-734 (1998)]. Arf was subsequently found to inhibit the negativecomponents of the p53-Mdm2 auto-regulatory loop by interfering withseveral of Mdm2's activities toward p53. First, by binding Mdm2, Arfinhibits Mdm2-dependent nucleo-cytoplasmic shuttling of p53 which leadsto stabilization and activation of p53 [Tao et al., Proc Natl Acad SciUSA, 96:6937-41(1999)]. Second, Arf inhibits the E3 ubiquitin ligaseactivity of Mdm2 toward p53 in vitro [Honda et al., Embo J, 18:22-7(1999); Midgley et al., Oncogene, 19:2312-23 (2000)] and is thought tobe an important aspect of Arf-dependent activation of p53 in vivo[Midgley et al., Oncogene, 19:2312-23 (2000); Llanos et al., Nat. CellBio., 3:445-452 (2001)]. Finally, Arf binds and sequesters Mdm2 in thenucleolus, physically separating Mdm2 and p53 in different sub-cellularcompartments [Weber et al., Nat. Cell Biol., 1:20-26 (1999); Lohrum etal., Nat. Cell Biol., 2:179-81 (2000); Weber et al., Mol. Cell Biol.,20:2517-2528 (2000)]. The relative importance of these three mechanismsto Arf-dependent stabilization and activation of p53 is a matter ofdebate. For example, a recent report shows that Mdm2 binding but notnucleolar localization is the functional property of Arf that isrequired for p53 activation [Llanos et al., Nat. Cell Biol., 3:445-452(2001)]. This report, however, does not rule out earlier reports thatnucleolar co-localization of Arf and Mdm2 contribute to p53stabilization through sequestration [Weber et al., Nat. Cell Biol.,1:20-26 (1999)]. It is likely that Arf acts via several mechanisms tostabilize p53 and these have evolved in concert with the multiplicity ofMdm2's effects on p53. Importantly, direct interaction between Arf andHdm2 is required for the multiple mechanisms of p53 stabilization.

[0010] Therefore, there is a need to further characterize the Arf-Hdm2complex. In addition, there is a need to determine the specific domainsof Arf and Hdm2 that are involved in this complex. Furthermore, there isa need to identify compounds that can mimic the effect of Arf on Hdm2,since the absence of functional Arf is commonplace in tumor cells.Alternatively, there is a need to identify compounds that inhibit thebinding of Arf to Hdm2 to prevent undesired activation of p53-dependentpathways by, for example, DNA damaging agents, in normal cells.

[0011] The citation of any reference herein should not be deemed as anadmission that such reference is available as prior art to the instantinvention.

SUMMARY OF THE INVENTION

[0012] Through disclosing that the binding of Arf with Hdm2 results inspecific domains of both proteins undergoing a dramatic transition fromdisordered conformations to extended structures comprised of β-strands,the present invention provides new insight towards theidentification/design of novel anti-cancer therapeutics. Thus, in aparticular aspect of the present invention unique assays are providedfor identifying compounds that mimic and/or enhance, or alternativelyinhibit the effect of Arf on Hdm2. In a related aspect of the presentinvention specific protein fragments derived from Arf and Hdm2 that playa direct role in the binding of these two important regulatory proteinsare provided.

[0013] Therefore, the present invention provides methods of identifyinga compound that can induce β-strand assembly of Dm2 (e.g., Hdm2 orMdm2). One such method comprises contacting the compound with Dm2 or aninducible fragment of Dm2 (e.g., a fragment of Dm2 that is capable ofbeing induced to β-strand assembly by Arf) and then determining whetherDm2 or the inducible fragment of Dm2 is induced to form a β-strandassembly by the compound. A compound is identified when Dm2 or theinducible fragment of Dm2 is induced to form a β-strand assembly. In aparticular embodiment, a peptide or protein comprising the Arf motif(i.e., the amino acid sequence of SEQ ID NO:13) responsible for inducingβ strand assembly in Hdm2, can be used as a positive control.

[0014] The present invention also provides methods of identifying acompound that can enhance the rate of β-strand assembly of Dm2 inducedby Arf. One such embodiment comprises contacting the compound with Dm2or an inducible fragment of Dm2, and Arf or an inducing fragment of Arfand then determining the rate of the β-strand assembly of Dm2 or of theinducible fragment of Dm2. A compound is identified that can enhance therate of β-strand assembly of Dm2 induced by Arf when the rate of theβ-strand assembly of Dm2 or of the inducible fragment of Dm2 increasesin the presence of the compound relative to in the absence of thecompound.

[0015] The present invention further provides methods of identifying acompound that can inhibit the formation of β-strand assembly of Dm2. Ina particular embodiment of this type the compound is contacted with Dm2or an inducible fragment of Dm2, and Arf or an inducing fragment of Arf,and the rate of formation of a β-strand assembly of Dm2 or the induciblefragment of Dm2 is determined. A compound is identified that can inhibitthe formation of β-strand assembly of Dm2 when the rate of formation ofthe β-strand assembly of Dm2 and/or the rate of formation of theβ-strand assembly of the inducible fragment of Dm2 decreases in thepresence of the compound relative to in its absence.

[0016] In a related embodiment, the compound is contacted with Dm2 or aninducible fragment of Dm2, and Arf or an inducing fragment of Arf, andthe amount of formation of a β-strand assembly of Dm2 or the induciblefragment of Dm2 is determined. A compound is identified that can inhibitthe formation of β-strand assembly of Dm2 when the amount of formationof the β-strand assembly of Dm2 or the inducible fragment of Dm2decreases in the presence of the compound relative to in its absence.

[0017] In a particular embodiment, the inducing and/or inhibiting ofβ-strand assembly of Dm2 or the inducible fragment of Dm2 is determinedby circular dichroism (CD) measurements. In another embodiment, theinducing and/or inhibiting of β-strand assembly of Dm2 or the induciblefragment of Dm2 is determined by nuclear magnetic resonance (NMR)measurements. In yet another embodiment, the inducing and/or inhibitingof β-strand assembly of Dm2 or the inducible fragment of Dm2 isdetermined by Fourier Transform Infra-red (FTIR) spectroscopy. In stillanother embodiment, the inducing and/or inhibiting of β-strand assemblyof Dm2 or the inducible fragment of Dm2 is determined by fluorescencespectroscopy. In another embodiment the natural fluorescence of one ormore tryptophan residues in Dm2 are used to monitor the induction and/orinhibition of β-strand assembly. In one such embodiment changes in theintensity, wavelength, and/or anisotropy of tryptophan emission are usedto monitor the binding of Arf to Dm2 and the formation of β-strandassemblies. In an alternative embodiment, a fluorescent probe, such asTexas Red™, is covalently bound to either Dm2 or Arf. Changes in thefluorescence intensity, excitation and/or emission wavelength, and/oranisotropty of the probe can be monitored when the unlabeled and labeledspecies are mixed together.

[0018] The present invention also provides methods of identifying acompound that can induce supramolecular assemblies comprised ofβ-strands of Dm2 or a inducible fragment of Dm2 (e.g., Hdm2 or Mdm2).One such method comprises contacting the compound with Dm2 or aninducible fragment of Dm2 that is capable of being induced to formsupramolecular assemblies by Arf, and then determining whether thecompound induces Dm2 or the inducible fragment of Dm2 to formsupramolecular assemblies. A compound is identified when Dm2 or theinducible fragment of Dm2 is induced to form supramolecular assemblies.In a particular embodiment, a peptide or protein comprising the Arfmotif, (i.e., the amino acid sequence of SEQ ID NO:13) can be used as apositive control. In one embodiment, the inducing of supramolecularassemblies of Dm2 or the inducible fragment of Dm2 is determined by sizeexclusion measurements. In a preferred embodiment of this type, theinducing of supramolecular assemblies of Dm2 or the inducible fragmentof Dm2 is determined by gel filtration chromatography.

[0019] In one embodiment, the Dm2 used in the methods of the inventionis Hdm2. In a preferred embodiment of this type, the Hdm2 comprises theamino acid sequence of SEQ ID NO:8. In still another embodiment, theinducible fragment of Hdm2 comprises amino acid residues 235-259 of SEQID NO:8, which is the H1 segment. In a related embodiment, the induciblefragment of Hdm2 comprises amino acid residues 275-289 of SEQ ID NO:8,which is the H2 segment. In a preferred embodiment, the induciblefragment of Hdm2 comprises both amino acid residues 235-259 and aminoacid residues 275-289 of SEQ ID NO:8.

[0020] The present invention also provides a compound that is identifiedby a method of the present invention. Preferably the compound does notcomprise five or more consecutive amino acids of a naturally occurringprotein. More preferably the compound is neither an amino acid nor madeup of animal acids (i.e., a compound which is not a peptide). Even morepreferably, the compound is a small molecule that that has a molecularweight of less than 3 Kilodaltons.

[0021] In related embodiments, compounds can be tested for their abilityto either enhance the effect of Arf or alternatively interfere with theformation of the Arf-Dm2 complex using similar protocols as outlinedabove except both Arf or an inducing fragment of Arf, and Dm2 or aninducible fragment of Dm2 are included in the assay.

[0022] The formation of supramolecular assemblies and/or β-strandassembly can be readily monitored, e.g., by NMR, CD, FTIR, fluorescenceand/or size exclusion. Therefore, a compound can be contacted with theArf and Dm2 (and/or fragments thereof) and the amount of formation ofthe supramolecular assemblies and/or β-strand assembly of the Arf-Dm2can be determined. When the compound decreases or eliminates thesupramolecular assemblies and/or β-strand assembly of the Arf-Dm2complex, the compound is identified as an inhibitor of the Arf-Dm2interaction. Similarly the kinetics of the rate of formation of thesupramolecular assemblies and/or β-strand assembly can be measured, andcompounds can be assayed to select inhibitors or enhancers of the rateof formation of the supramolecular assemblies and/or β-strand assembly,as exemplified herein.

[0023] All of the methods for identifying compounds of the presentinvention can be performed by adding a compound to the assay solution atany time during the assay, including making additions at multiple times.Thus the compound can be added: (i) prior to the addition of Arf and/oran inducing fragment of Arf; and/or (ii) prior to the addition of Dm2and/or an inducible fragment of Dm2; and/or (iii) together with Arfand/or an inducing fragment of Arf; and/or (iv) together with Dm2 and/oran inducible fragment of Dm2; and/or (v) after the addition of Arfand/or an inducing fragment of Arf; and/or (vi) after the addition ofDm2 and/or an inducible fragment of Dm2.

[0024] In addition the present invention provides methods of designingcompounds that are predicted to mimic, enhance or alternatively inhibitthe Arf-induced formation of β-strand assembly of Dm2. One such methodcomprises defining the structure of the Arf-Dm2 complex by usingcomputer-based molecular modeling and docking techniques. In thisapproach, ensembles of molecular models for segments of Arf (forexample, the Arf motif, or the segments of human or mouse Arf embodiedby this motif) and Dm2 (for example, the H1 and/or H2 segments) aregenerated that are consistent with CD and FT-IR spectra for the Arf-Dm2complex, namely that the polypeptide backbone torsion angles adoptvalues allowed in β-strands. Then, each member of the Arf ensemble issystematically docked with each member of the Dm2 ensemble usingprograms such as DOCK, or AUTODOCK. During the docking stage of theprocedure, the binding energy for each of a large number of alternativeArf-Dm2 binding configurations is calculated and the dockedconfigurations ranked according to binding energy. The Arf-Dm2 bindingmodels with the lowest overall binding energy will then be used todesign and/or identify a compound that is predicted to mimic, enhance,or alternatively inhibit the Arf-induced formation of β-strand assemblyof Dm2.

[0025] As the skilled artisan would readily recognize, compoundsdesigned and/or identified by this method can then be synthesized (ifnecessary) and tested in any of a number of assays, including thosedescribed above. For example, in one such embodiment the method furthercomprises contacting the compound with Dm2 or an inducible fragment ofDm2 and then determining whether Dm2 or the inducible fragment of Dm2 isinduced to form a β-strand assembly. The compound is identified as amimic of Arf if Dm2 or the inducible fragment of Dm2 is induced to forma β-strand assembly.

[0026] The present invention further provides methods of treatingpatients with cancer and/or patients having a predisposition fordeveloping cancer. In a particular embodiment of this type, the patienthas a tumor with cells that are characterized by a lack of sufficientArf activity (e.g., lacking of a functional Arf), but still retainfunctional p53. One specific embodiment comprises administering to apatient a compound that mimics and/or enhances Arf activity that wasidentified by a method of the present invention. In a relatedembodiment, a compound is administered that can induce β-strand assemblyof Dm2 in a cell that is lacking a functional Arf protein and/orsufficient Arf activity to de-repress the repression of p53 mediatedapoptosis by Dm2 and thereby arrest cell growth.

[0027] The present invention also provides specific fragments of the Arfand Dm2 proteins and peptides comprising the amino acid sequences ofsuch fragments that can induce β-strand assembly of Dm2. Fusion proteins(including chimeric proteins) comprising these fragments/peptides arealso provided, as are nucleic acids encoding such fragments/peptides,and corresponding fusion proteins. Preferably the fragments/peptides arebetween 8 and 50 amino acids in length. In one such embodiment thefragment/peptide comprises and/or consists of the amino acid sequence ofSEQ ID NO:13. In a particular embodiment of this type, thefragment/peptide comprises and/or consists of the amino acid sequence ofSEQ ID NO:9. In a another embodiment of this type, the fragment/peptidecomprises and/or consists of the amino acid sequence of SEQ ID NO:10. Inyet another embodiment, the fragment/peptide comprises and/or consistsof the amino acid sequence of SEQ ID NO:11. In still another embodimentof this type, the fragment/peptide comprises and/or consists of theamino acid sequence of SEQ ID NO:12.

[0028] In a preferred embodiment the fragment/peptide comprises oralternatively consists of two or more segments of the Arf protein eachsegment comprising and/or consisting of the amino acid sequence of SEQID NO:13. In one embodiment of this type, the fragment/peptide comprisesand/or consists of both the amino acid sequence of SEQ ID NO:9 and SEQID NO:10. In another embodiment, the fragment/peptide comprises and/orconsists of both the amino acid sequence of SEQ ID NO:11 and SEQ IDNO:12. In still another embodiment, the fragment/peptide comprisesand/or consists of both the amino acid sequence of SEQ ID NO:9 and SEQID NO:12. In yet another embodiment, the fragment/peptide comprisesand/or consists of both the amino acid sequence of SEQ ID NO:10 and SEQID NO:11.

[0029] In addition to peptides comprising the Arf segments describedabove, the invention also provides compositions comprised of at leastone pair of the peptide segments described above linked together by anon-peptide linkage, e.g., a non-peptide chemical linkage.

[0030] In a related embodiment, the present invention provides afragment/peptide that comprises and/or consists of amino acid residues235-259 of SEQ ID NO:8. In still another embodiment of this type thefragment/peptide comprises and/or consists of amino acid residues275-289 of SEQ ID NO:8. In a preferred embodiment, the fragment/peptidecomprises and/or consists of amino acid residues 235-259 and amino acidresidues 275-289 of SEQ ID NO:8.

[0031] In another aspect of the present invention, antibodies raisedagainst specific fragments/peptides of Arf and/or Dm2 are provided. Inone such embodiment the antibody is raised against a fragment/peptidecomprising an amino acid sequence of SEQ ID NO:13. In anotherembodiment, the antibody is raised against a fragment/peptide comprisingamino acid residues 235-259 of SEQ ID NO:8. In yet another embodiment,the antibody is raised against a fragment/peptide comprising amino acidresidues 275-289 of SEQ ID NO:8. In a preferred embodiment, the antibodyis raised against a fragment/peptide comprising amino acid residues235-259 and amino acid residues 275-289 of SEQ ID NO:8. In oneembodiment, the antibody is a polyclonal antibody. In anotherembodiment, the antibody is a monoclonal antibody. In still anotherembodiment, the antibody is a chimeric and/or humanized antibody.

[0032] The present invention further provides methods of inducingapoptosis in a cell. In one such embodiment, apoptosis is induced byadministering an antibody of the present invention to a cell. In apreferred embodiment of this type the antibody is a humanized antibody.In a related embodiment, apoptosis is induced by administering acompound identified by a method of the present invention to the cell.

[0033] The present invention further provides methods of treating apatient for which induced apoptosis in targeted cells is a desirabletreatment, such as various forms of cancer in which p53 is functionaland Dm2 is overexpressed and/or p53 is functional and Arf is notfunctional. One or both of these conditions is thought to be acontributing factor in the development of a wide variety of cancers,including acute myeloid leukemia [Faderl, et. al. Cancer, 89:1976-82(2000) ], breast cancer [Takami etal. Breast Cancer, 30: 95-102 (1994)],Burkitt lymphoma [Lindstrom et. al. Oncogene, 20: 2117-7 (2001)], clearcell renal cell carcinoma [Haitel et. al. Clin. Cancer Res., 6: 1840-4(2000)], colon carcinomas [Burri et. al. Lab. Invest., 81: 217-29(2001)], ependymomas [Suzuki and Iwaki Mod. Pathol. 13:548-53 (2000)],gastric cancer [Villaseca et. al. Rev. Med. Chil., 128:127-36 (2000)],glioblastoma [Fulci et. al. Oncogene, 19: 3816-22 (2000)], Hodgkin'sdisease [Kupper et. al. Br. J. Haematol., 112: 768-75 (2001)],intrahepatic cholangiocarcinoma [Horie. Archet. al. Virchows., 437:25-30 (2000)], intimal sarcomas arising in the pulmonary artery[Bode-Lesniewska et. al. Virchows. Arch., 438: 57-65 (2001)], malignantpleural mesothelioma [Yang et. al. Cancer Res., 61: 5959-63 (2001)],melanoma-neural system tumour syndrome [Randerson-Moor et. al. Hum.Molec. Genet., 10: 55-62 (2001)], non-Hodgkin's lymphomas [Pagnano et.al. Am. J. Hematol., 67: 84-92 (2001)], non-small cell lung carcinomas[Gorgoulis et. al. Mol. Med., 6: 208-37 (2000)], ovarian tumors [Palazzoet. al. Hum. Pathol., 31: 698-704 (2000)], oral cancer [Ralhan et. al.Am. J. Pathol., 157: 587-96 (2000)], oral squamous cell carcinoma [Sanoet. al. Pathol. Int., 50: 709-16 (2000)], paragangliomas [Lam et. al. J.Clin. Pathol., 54: 443-8 (2001)], phaeochromocytomas [Lam et. al. J.Clin. Pathol., 54: 443-8 (2001)], primary central nervous systemlymphomas [Nakamura et. al. Cancer Res., 61 6335-39 (2001)], prostatecarcinoma [Leite et. al. Mod. Pathol. 14: 428-36 (2001)], soft tissuesarcoma [Bartel et. al. Int. J. Cancer, 95:168-75 (2001)], and urinarybladder carcinoma [Ioachim et. al. Histol. Histopathol., 15: 721-7(2000)].

[0034] One such method comprises administering an antibody of thepresent invention to a patient. Preferably, the treatment isadministered to a patient that has a tumor containing cellscharacterized by the presence of functional Hdm2, and functional p53.

[0035] The present invention also provides kits for identifying acompound that can induce β-strand assembly of Dm2 in the absence and/orpresence of Arf. One such kit comprises a fragment of Hdm2 thatcomprises amino acid residues 235-259 of SEQ ID NO:8. In anotherembodiment, the kit comprises a fragment of Hdm2 that comprises aminoacid residues 275-289 of SEQ ID NO:8. In a particular embodiment the kitcomprises a fragment of Hdm2 that comprises amino acid residues 235-259and amino acid residues 275-289 of SEQ ID NO:8. In another embodiment apeptide that comprises the amino acid sequence of SEQ ID NO:13 is alsoincluded. Preferably this peptide comprises two copies of the amino acidsequence of SEQ ID NO:13. More preferably the kit further comprisesinstructions for identifying a compound that can induce β-strandassembly of Dm2.

[0036] Accordingly, it is a principal object of the present invention toprovide an assay for selecting drugs that can be used to treat cancer.

[0037] It is a further object of the present invention to provide agentsthat can mimic and/or enhance the ability of Arf to stimulate β-strandassembly of Dm2.

[0038] It is a further object of the present invention to providepeptides consisting of defined minimal domains of Arf and Dm2 that arenecessary and sufficient for Arf-Dm2 binding.

[0039] It is a further object of the present invention to providemethods of treating diseases that are adversely affected by theexistence of cells that do not contain sufficient Arf activity.

[0040] It is a further object of the present invention to providemethods of identifying agents that can interfere with the ability of Arfto bind Dm2, including antibodies to Arf or Dm2.

[0041] It is a further object of the present invention to provide agentsthat can interfere with the ability of Arf to bind Dm2.

[0042] It is a further object of the present invention to provideantibodies that can interfere with the ability of Arf to bind Dm2.

[0043] These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE SEQUENCES

[0044] SEQ ID NO: TYPE ORGANISM PROTEIN  1 Nucleic Acid Mouse Arf  2Amino Acid Mouse Arf  3 Nucleic Acid Human Arf  4 Amino Acid Human Arf 5 Nucleic Acid Mouse Mdm2  6 Amino Acid Mouse Mdm2  7 Nucleic AcidHuman Hdm2  8 Amino Acid Human Hdm2  9 Amino Acid Mouse mA1 10 AminoAcid Mouse mA2 11 Amino Acid Human hA1 12 Amino Acid Human hA2 13 AminoAcid Consensus Arf Motif 14 Amino Acid Consensus RRPR 15 Amino AcidHuman N-terminal 1-37 of Arf 16 Amino Acid Mouse N-terminal 1-37 of Arf17 Amino Acid Opossum N-terminal 1-37 of Arf 18 Amino Acid Human˜210-304 of Dm2 19 Amino Acid Mouse ˜210-304 of Dm2 20 Amino AcidHamster ˜210-304 of Dm2 21 Amino Acid Horse ˜210-304 of Dm2 22 AminoAcid Dog ˜210-304 of Dm2 23 Amino Acid Chicken ˜210-304 of Dm2 24 AminoAcid Zebrafish ˜210-304 of Dm2 25 Amino Acid Tree Frog ˜210-304 of Dm2

BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIGS. 1a-1 b show the surface plasmon resonance sensograms of Hdm2constructs binding to mArfN37. His-tagged mArN37 was immobilized on theSPR biosensor surface using a covalently linked His antibody. Binding ofHdm2 210-304 of SEQ ID NO:8 (FIG. 1a) and Hdm2 210-275 of SEQ ID NO:8(FIG., 1 b).

[0046]FIGS. 2a-2 c show the structure prediction for Arf and Hdm2binding domains. Alignment of sequences for residues 1-37 of human (SEQID NO:15), mouse (SEQ ID NO:16) and opossum (SEQ ID NO:17) Arf (FIG. 2a)and ˜ residues 210-304 of Mdm2 (FIG. 2b) from several species listed inTHE BRIEF DESCRIPTION OF THE SEQUENCES above, corresponding to SEQ IDNOs:18-25. Residues that are underlined are conserved in all sequencesand those in bold type are conserved in several sequences. The programJnet was used to predict secondary structure and solvent exposure withinthe aligned regions. The secondary structure predictions are labeled“Jnet pred.”; β-strand secondary structure is abbreviated “E”, α-helix“H”, and random coil “−”. The prediction confidence score is labeled“Jnet conf.”, with 0 the lowest and 9 the highest confidence values. Theprediction of solvent exposure is labeled “Solv. Exp.”, with “B”indicating that a residue is predicted to be less than 25% solventexposed and “−” indicating greater than 25% solvent exposure. Theschematic illustration of peptides derived from Hdm2 210-304 that bindmArfN37 is shown in FIG. 2c.

[0047]FIG. 3 shows the characterization of Arf:Hdm2 assemblies. CDspectra of mArfN37 complexed with Hdm2 210-304 (solid line) and Hdm2210-275 (dotted line) are consistent with β-strand secondary structure.The molar ratio of the two species in each sample is given.

[0048]FIG. 4 shows the “Arf motif” (consensus sequence, SEQ ID NO:13). Ashort sequence of 8 or 9 amino acids is repeated twice in mouse andhuman Arf. An alternating hydrophobic/charge pattern mediates binding.

[0049]FIGS. 5a-5 c show the surface plasmon resonance (SPR) bindingexperiments with Arf and Hdm2 peptides which reveal sites ofinteraction. His-tagged Hdm2 210-304 (FIGS. 5a-5 b) and His-taggedmArfN37 (FIG. 5c) were captured on the SPR surface with an anti-Hisantibody. The binding of peptides derived from the N-terminus of mouseArf (FIG. 5a), human Arf (FIG. 5b), or the central, acidic domain ofHdm2 (FIG. 5c) was monitored. *, this peptide is anomalous because itbinds extensively to the reference cell.

DETAILED DESCRIPTION OF THE INVENTION

[0050] Isolated Arf and Hdm2 domains are dynamically disordered insolution, yet they retain the ability to interact in vitro and incellular assays. As shown below, upon binding, domains of both Arf andHdm2 undergo a dramatic transition from disordered conformations toextended structures comprised of β-strands. The presence of domains fromboth proteins is necessary and sufficient for the formation of thehighly stable extended α structures. Sites within Arf and Hdm2 thatinteract at a resolution of 5 amino acids have been mapped using surfaceplasmon resonance (SPR). SPR and circular dichroism (CD)spectropolarimetry confirm the presence of multiple interaction domainswithin each protein (see Example below).

[0051] As disclosed herein, small peptide segments are identified withinArf and Hdm2 that are responsible for the interactions of these twoproteins and mediate their nucleolar localization. Furthermore, pure Arfand Hdm2 are both shown to be dynamically disordered in solution butthat, when mixed in vitro, they adopt highly stable β-sheet structures.The β-structures prepared in vitro, however, are extended networks andare relevant to the structures that form when Arf and Hdm2 interact incells within the nucleoplasm and/or nucleoli.

[0052] Both p14^(Arf) (human) and p19^(Arf) (mouse) interact with Hdm2through two short motifs present in their N-termini. The Arf interactingregion of Hdm2 is also composed of two short sequences located in thecentral acidic domain, between residues 235-264 and 270-289 of SEQ IDNO:8. The binding-induced structural transition is also induced by shortpeptides, 15 amino acids in length, which contain the binding motifs.Micro-injection and live cell imaging of proteins tagged withfluorescent labels was used to confirm the in vivo function of theinteraction domains. Arf and Hdm2 thus appear to interact through anovel mechanism that exerts control over the cell division cycle. Adetailed analysis of Arf/Hdm2 interactions is disclosed herein. Thepresent invention therefore provides unique opportunities for thedevelopment of anticancer therapeutics due to the novel interactionbetween Dm2 and Hdm2 and the limited size of the protein domainsinvolved.

[0053] Furthermore, small segments of Arf and Hdm2 have been identifiedthat mediate binding and that can play a role in regulating Hdm2'srepressor function toward p53. Using this information, it is nowpossible to inhibit p53 destruction by disrupting inter-domaininteractions within Hdm2 with molecules that mimic and/or enhance Arffunction. The Arf motif is relatively small, and may be mimicked by yetsmaller molecules. Similarly, the molecular targets of this motif, theH1 and H2 segments of Hdm2, are small. These findings provide a methodfor searching for small molecules that bind Hdm2 in a manner that mimicsArf, and that may produce biological effects similar to those producedby Arf. Since many human cancers are characterized by Arf loss while p53is maintained in wild-type form [Sherr, Cancer Res., 60: 3689-95(2000)], this methodology should have wide-ranging implications in thetreatment of cancer in humans. Abbreviations: BrdU 5-bromodeoxyuridineCD circular dichroism CHAPSO 3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonic acid DAPI 4′-6-Diamidino-2-phenylindole-2HCl DMEMDulbecco's modified Eagle's medium EDCN-Ethyl-N′-(3-Dimethylaminopropyl)Carbodiimide FBS fetal bovine serumFMOC- α-(9-fluorenylmethyloxycarbonyl)-amino acid GFP green fluorescentprotein HBS-N 0.01 M HEPES, pH 7.4, 0.15 M NaCl buffer HBTU2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate HEPES 4-(2-Hydroxyethyl)-1-PiperazineethanesulfonicAcid HMP hydroxymethylphenyl-polystyrene resin HOBtN-hydroxybenzotriazole NHS N-Hydroxysuccinimide NLS nuclear localizationsignal NMR nuclear magnetic resonance NoLS nucleolar localization signalPBS phosphate-buffered saline SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis SPR surface plasmon resonance TFAtrifluoroacetic acid

[0054] Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0055] As used herein the terms “Arf”, “ARF”, “p19^(ARF) protein,”“p19^(ARF)”, “ARF-p19”, “ARF-p19/ARF-p14”, “p14^(ARF) protein,”“p14^(ARF)”, or “ARF-p14” are all used interchangeably except that“p14^(ARF) protein,” “p14^(ARF)”, “ARF-p14” in general referspecifically to the human protein. Arf is involved in regulation of theeukaryotic cell cycle. The Arf protein is encoded by a nucleic acidderived from the gene locus, INK4A-Arf, which also encodes an inhibitorof D-type cyclin-dependent kinases termed “p16^(InK4a) protein,”“p16^(InK4)” or simply “InK4a-p16.” [see also Quelle et al., Cell,83:993-1000 (1995)]

[0056] An “active fragment” of an Arf protein is a peptide orpolypeptide that comprises a fragment of Arf and retains at least onephysiological activity of the Arf e.g. by acting as a tumor suppressorand/or having the ability to bind to Dm2. Examples of active fragmentsof Arf are the peptides encoded by exon 1β, e.g. amino acid residues1-62 of SEQ ID NO:4 and the peptide encoded by amino acid residues 1-37of SEQ ID NO:2. A fusion protein comprising an active fragment of an Arfprotein can often be used interchangeably with an active fragment of anArf protein, and such fusion proteins are meant to be included when theterm “active fragment” of an Arf protein is used.

[0057] As used herein a peptide “consisting of a minimal domain of Arf”is a peptide comprising the minimum portion of the full-length Arf thatstill retains the ability of the full-length Arf protein to bind Dm2 andact as a tumor repressor.

[0058] As used herein, an “inducing fragment” of an Arf protein is anactive fragment of an Arf protein that can induce supramolecularassemblies and/or β-strand assembly of (i) DM2; and/or (ii) an induciblefragment of Dm2; and/or (iii) an Arf-Dm2 complex. Preferably an inducingfragment of Arf comprises two copies of the Arf motif each comprisingthe amino acid sequence of SEQ ID NO:13. A fusion protein comprising aninducing fragment of an Arf protein can often be used interchangeablywith an inducing fragment of an Arf protein, and such fusion proteinsare meant to be included when the term inducing fragment of an Arfprotein is used.

[0059] As used herein the term “sufficient Arf activity” means activitysufficient to arrest the entry of cells into the cell division cycle,which is a hallmark of Arf activity. This activity has beencharacterized [Weber, et al., Nat. Cell Biol. 1:20-26 (2000); Weber, etal., Mol. Cell Biol. 20:2517-2528 (2000) and U.S. application Ser. No.09/480,718, filed Jan. 7, 2000, the contents of which are herebyincorporated by reference in their entireties]. Further, this activityhas been characterized for a fragment of mouse p19^(Arf) containingresidues 1-37 [DiGiammarino, et al., Biochemistry 40:2379-2386 (2001),the contents of which are hereby incorporated by reference in theirentireties].

[0060] The abbreviation “DM2” or “Dm2” as used herein refers to thegeneric form of the protein “Mdm2” and its human ortholog “Hdm2” whichare Murine Double Minute 2 and Human Double Minute 2 respectively. Hdm2has the GenBank accession number of M92424, an amino acid sequence ofSEQ ID NO:8 and a nucleic acid sequence of SEQ ID NO:7. Mdm2 has theGenBank accession number of X58876, an amino acid sequence of SEQ IDNO:6 and a nucleic acid sequence of SEQ ID NO:5. Mdm2, for example, canbind to the N-terminal transcriptional activation domain of p53 to blockexpression of p53-responsive genes [Momand et al., Cell 69:1237-1245(1992); Oliner et al., Nature 362:857-860 (1993)], it has an intrinsicE3 ligase activity that conjugates ubiquitin to p53 [Honda and Yasuda,Oncogene 19:1473-1476 (2000)] and it also appears to play a role inshuttling p53 from the nucleus to the cytoplasm, where p53 is degradedin cytoplasmic proteasomes [Freedman and Levine, Mol. Cell. Biol.18:7288-7293 (1998); Roth et al., EMBO J. 17:554-564 (1998); Tao andLevine, Proc. Natl. Acad. Sci. 96:3077-3080 (1999)].

[0061] As used herein a peptide “consisting of a minimal domain of Dm2”is a peptide comprising the minimum portion of the full-length Dm2 thatstill retains the ability to bind Arf and thereby competitively inhibitthe binding of Arf with the full-length DM2.

[0062] As used herein, an “inducible fragment” of a Dm2 protein is afragment of a Dm2 protein that can be induced to form supramolecularassemblies and/or β-strand assemblies by Arf, and/or an inducingfragment of Arf. An inducible fragment of Dm2 may be part of an Arf-Dm2complex. Preferably an inducible fragment of Dm2 comprises the aminoacid residues 235-259 of SEQ ID NO:8 and/or the amino acid residues275-289 of SEQ ID NO:8. A fusion protein comprising an induciblefragment of a Dm2 protein can often be used interchangeably with aninducible fragment of a Dm2 protein, and such fusion proteins are meantto be included when the term an inducible fragment of a Dm2 protein isused.

[0063] As used herein the terms “fusion protein” and “fusion peptide”are used interchangeably and encompass “chimeric proteins and/orchimeric peptides”. A fusion protein comprises at least a portion of oneprotein such as ARF-p19 joined via a peptide bond to at least anotherportion of a protein or peptide that it is not naturally contiguouslyconnected to. For example, a fusion peptide of the present inventionincludes a peptide that consists of two consecutive nonamers and/oroctamers each having the amino acid sequence of SEQ ID NO:13. In anotherembodiment, the fusion peptide can comprise amino acid residues of SEQID NO:11 that is covalently joined to a linker peptide which in turn isbound to amino acid residues of SEQ ID NO:12. Fusion proteins andpeptides can also, and/or alternatively comprise a marker protein orpeptide as exemplified below, or a protein or peptide that aids in theisolation and/or purification of the fusion protein.

[0064] A “heterologous nucleotide sequence” as used herein is anucleotide sequence that is added to a nucleotide sequence of thepresent invention by recombinant methods to form a nucleic acid which isnot naturally formed in nature. Such nucleic acids can encode fusion(e.g. chimeric) proteins. Thus the heterologous nucleotide sequence canencode peptides and/or proteins which contain regulatory and/orstructural properties. In another such embodiment the heterologousnucleotide sequence can encode a protein or peptide that functions as ameans of detecting the protein or peptide encoded by the nucleotidesequence of the present invention after the recombinant nucleic acid isexpressed. In still another embodiment the heterologous nucleotidesequence can function as a means of detecting a nucleotide sequence ofthe present invention. A heterologous nucleotide sequence can comprisenon-coding sequences including restriction sites, regulatory sites,promoters and the like.

[0065] As used herein a “polypeptide” is used interchangeably with theterm “protein” and denotes a polymer comprising two or more amino acidsconnected by peptide bonds. Preferably, a polypeptide is furtherdistinguished from a “peptide” with a peptide comprising about twenty orless amino acids, and a polypeptide or protein comprising more thanabout twenty amino acids. Preferably a protein fragment is defined as apeptide or a polypeptide employing the same size criteria.

[0066] As used herein “supramolecular assemblies comprised of β-strands”describes peptides or polypeptides that bind together to form highmolecular weight assemblies. In the present case, Arf and Dm2 bindtogether to form assemblies comprised of β-strands. These assemblies arecomprised of many molecules of Arf and Dm2. The molecular size of theseassemblies is characterized using, for example, gel filtrationchromatography, wherein the assemblies elute at early times in theexcluded volume and appear to have a molecular weight of 200Kilodaltons, or greater.

[0067] As used herein a “small organic molecule” is an organic compound[or organic compound complexed with an inorganic compound (e.g., metal)]that has a molecular weight of less than 3 Kilodaltons, and preferablyless than 1.5 Kilodaltons. A “compound” of the present invention ispreferably a small organic molecule. Preferably, the small organicmolecules identified by the methods of the present invention are notpeptides.

[0068] As used herein the terms “solid substrate” and “solid support”are used interchangeably and represent a solid material that provides aninert surface that allows a biological reaction to be performed. Solidsupports include biological chip plates as exemplified by Rava et al.,U.S. Pat. No. 5,874,219, the contents of which are hereby incorporatedby reference in their entireties and multi-well (multi-titer) quartz andpolystyrene plates. Examples of material that can be used as solidsubstrates include glass, peptide polymers (e.g., collagen), peptoidpolymers, polysaccharides (including commercial beads, e.g., SEPHADEXand the like), carbohydrates, hydrophobic polymers, polymers, tissueculture polystyrene, metals, derivatized plastic films, glass beads,plastic beads, alumina gels, magnetic beads, nitrocellulose, cellulose,and nylon membranes.

[0069] A molecule is “antigenic” when it is capable of specificallyinteracting with an antigen recognition molecule of the immune system,such as an immunoglobulin (antibody) or T cell antigen receptor. Anantigenic polypeptide contains at least about 5, and preferably at leastabout 10, amino acids. An antigenic portion of a molecule can be thatportion that is immunodominant for antibody or T cell receptorrecognition, or it can be a portion used to generate an antibody to themolecule by conjugating the antigenic portion to a carrier molecule forimmunization. A molecule that is antigenic need not be itselfimmunogenic, i.e., capable of eliciting an immune response without acarrier.

[0070] The phrase “pharmaceutically acceptable” refers to molecularentities and compositions that are physiologically tolerable and do nottypically produce an allergic or similar untoward reaction, such asgastric upset, dizziness and the like, when administered to a human.Preferably, as used herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the compound is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water or aqueous solution salinesolutions and aqueous dextrose and glycerol solutions are preferablyemployed as carriers, particularly for injectable solutions. Suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

[0071] The phrase “therapeutically effective amount” is used herein tomean an amount sufficient to reduce by at least about 15 percent,preferably by at least 50 percent, more preferably by at least 90percent, and most preferably prevent, a clinically significant deficitin the activity, function and response of the host. Alternatively, atherapeutically effective amount is sufficient to cause an improvementin a clinically significant condition/symptom in the host, i.e., ashrinkage of a tumor.

[0072] As used herein, the term “ortholog” refers to the relationshipbetween proteins that have a common evolutionary origin and differbecause they originate from different species or strains. For example,mouse ARF-p19 is an ortholog of human ARF-p14.

[0073] As used herein an amino acid sequence is 100% “homologous” to asecond amino acid sequence if the two amino acid sequences areidentical, and/or differ only by neutral or conservative substitutionsas defined below. Accordingly, an amino acid sequence is 50%“homologous” to a second amino acid sequence if 50% of the two aminoacid sequences are identical, and/or differ only by neutral orconservative substitutions.

[0074] As used herein, DNA and protein sequence percent identity can bedetermined using MacVector 6.0.1, Oxford Molecular Group PLC (1996) andthe Clustal W algorithm with the alignment default parameters, anddefault parameters for identity. These commercially available programscan also be used to determine sequence similarity using the same oranalogous default parameters.

[0075] Polypeptides, peptides, or protein fragments of the presentinvention include, but are not limited to, those containing part of theamino acid sequences of an Arf protein and/or an Dm2 protein, includingaltered sequences in which functionally equivalent amino acid residuesare substituted for residues within the sequence resulting in aconservative amino acid substitution. Such alterations define the term“a conservative substitution” as used herein. For example, one or moreamino acid residues within the sequence can be substituted by anotheramino acid of a similar polarity, which acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Amino acids containingaromatic ring structures are phenylalanine, tryptophan, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point.

[0076] Particularly preferred conservative substitutions are:

[0077] Lys for Arg and vice versa such that a positive charge may bemaintained;

[0078] Glu for Asp and vice versa such that a negative charge may bemaintained;

[0079] Ser for Thr such that a free —OH can be maintained; and

[0080] Gln for Asn such that a free NH2 can be maintained.

[0081] As used herein the term “approximately” is used interchangeablywith the term “about” and signifies that a value is within twentypercent of the indicated value i.e., a protein containing“approximately” 50 amino acid residues can contain between 40 and 60amino acid residues.

Candidate Compounds

[0082] A candidate compound can be obtained by a number of means,including from a commercially available chemical library or an “inhouse” pharmaceutical library.

[0083] Examples of libraries of compounds that are commerciallyavailable include the Available Chemicals Directory (ACD,) the Specs andBioSpecs database, the Maybridge database, and the Chembridge database.Examples of pharmaceutical companies with “in house” chemical librariesinclude Merck, GlaxoSmithKline, Bristol Myers Squibb, Eli Lilly,Novartis, and Pharmacia.

[0084] Alternatively, candidate compounds can also be synthesized denovo either individually or as combinatorial libraries [Gordon et al.,J. Med. Chem. 37:1385-1401(1994)]. They may also be obtained from phagelibraries. Phage libraries have been constructed which when infectedinto host E. coli produce random peptide sequences of approximately 10to 15 amino acids [Parmley and Smith, Gene 73:305-318 (1988); Scott andSmith, Science 249:386-390 (1990)]. Once a phage encoding a peptide thatcan act as a potential drug has been purified, the sequence of thepeptide contained within the phage can be determined by standard DNAsequencing techniques. Once the DNA sequence is known, syntheticpeptides can be generated which are encoded by these sequences.

[0085] Since the present invention discloses the critical portions ofthe mutual binding domains of Arf and Dm2, the bound Arf-Dm2 peptidebinding complex can be readily analyzed to determine theirthree-dimensional structure. Using this structural information,potential mimics for the Arf peptides or inhibitors of the Arf-Dm2binding can be examined through the use of computer modeling using adocking program such as DOCK, GRAM, or AUTODOCK [Dunbrack et al.,Folding & Design, 2;27-42 (1997)]. This procedure can include computerfitting of candidate compounds to Dm2 for example, to determine how wellthe shape and the chemical structure of the candidate compound can bindto the Dm2 fragment [Bugg et al., Scientific American, December:92-98(1993); West et al., TIPS 16:67-74 (1995)]. Computer programs can alsobe employed to estimate the attraction, repulsion, and steric hindranceof the Arf or Dm2 peptides with a candidate compound.

[0086] Generally, the greater the steric complementarity and the greaterthe attractive forces, the more potent the candidate compound sincethese properties are consistent with a tighter binding constant.Furthermore, the more specificity in the design of a candidate compound,the more likely that the resulting drug will not interact as well withother proteins. This will minimize potential side-effects due tounwanted interactions with other proteins.

[0087] Systematic modification of selected compounds by computermodeling programs can then be performed until one or more compounds areidentified. Such analysis has been shown to be effective in thedevelopment of HIV protease inhibitors [Lam et al, Science 263: 380-384(1994); Wlodawer et al, Ann. Rev. Biochem. 62:543-585 (1993); Appelt,Perspectives in Drug Discovery and Design 1:23-48 (1993); Erickson,Perspectives in Drug Discovery and Design 1:109-128 (1993)].

[0088] In another such example, Selzer et al. [Exp. Parasitol.87(3):212-221 (1997)] screened the Available Chemicals Directory (adatabase of about 150,000 commercially available compounds) forpotential cysteine protease inhibitors, using DOCK3.5. Based on bothsteric and force field considerations, they selected 69 compounds. Ofthese, three had IC50's below 50 μM (i.e., the concentration of thecompound required to inhibit the reaction rate by 50%)

[0089] In addition, amino acid analogs, or peptidomimetics can be usedthat employ one or more unnatural or synthetic amino acids, such asusing a D amino acid. The subunits may be linked by peptide bonds or byother the bonds, e.g., an ester, ether, etc. A good starting point fordesigning such a peptidomimetic is of course a peptide of the presentinvention, e.g., one comprising the amino acid sequence of SEQ ID NO:13.

[0090] Synthetic peptides prepared using the well known techniques ofsolid phase, liquid phase, or peptide condensation techniques, or anycombination thereof, can thus include natural and unnatural amino acids.Amino acids used for peptide synthesis may be standard Boc (N-aminoprotected N-t-butyloxycarbonyl) amino acid resin with the standarddeprotecting, neutralization, coupling and wash protocols of theoriginal solid phase procedure of Merrifield [J. Am. Chem. Soc.85:2149-2154 (1963)], or the base-labile N-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han [J. Org. Chem. 37:3403-3409 (1972)]. Both Fmoc and BocN^(α)-amino protected amino acids can be obtained from Fluka, Bachem,Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, orPeninsula Labs or other chemical companies familiar to those whopractice this art. In addition, the method of the invention can be usedwith other N-protecting groups that are familiar to those skilled inthis art. Solid phase peptide synthesis may be accomplished bytechniques familiar to those in the art and provided, [for example, inStewart and Young Solid Phase Synthesis, Second Edition, Pierce ChemicalCo., Rockford, IL (1984); and Fields and Noble, Int. J. Pept. ProteinRes. 35:161-214 (1990)], or using automated synthesizers, such as soldby ABS. Thus, polypeptides of the invention may comprise D-amino acids,a combination of D- and L-amino acids, and various “designer” aminoacids (e.g., β-methyl amino acids, C α-methyl amino acids, andN-α-methyl amino acids, etc.) to convey special properties. Syntheticamino acids include ornithine for lysine, fluorophenylalanine forphenylalanine, and norleucine for leucine or isoleucine. Additionally,by assigning specific amino acids at specific coupling steps, α-helices,β turns, β sheets, γ-turns, and cyclic peptides can be generated.

[0091] In one aspect of the invention, the peptides may comprise aspecial amino acid at the C-terminus which incorporates either a CO₂H orCONH₂ side chain to simulate a free glycine or a glycine-amide group.Another way to consider this special residue would be as a D or L aminoacid analog with a side chain consisting of the linker or bond to thebead. In one embodiment, the pseudo-free C-terminal residue may be ofthe D or the L optical configuration; in another embodiment, a racemicmixture of D and L-isomers may be used.

[0092] In any case, compounds can be selected, for example, for theirability to induce β-strand formation of the Hdm2 fragments disclosedherein. A “lead” compound can then be identified for use as a focus of adrug development project.

Methods of Identifying Compounds that Affect the Arf-Dm2 Interaction

[0093] As disclosed by the present invention, new β-strand secondarystructure forms when Arf and Hdm2 interact. This β-strand secondarystructure is due to the interaction of small, specific domains presentin Arf (e.g., the Arf motif having the amino acid sequence of SEQ IDNO:13) and Dm2 (e.g., H1 and H2 as defined in the example below). Inaddition, as disclosed herein, the binding of Arf and Dm2 does not leadto a bimolecular complex as expected, but rather involves large,extended structures with predominantly β-strand secondary structure,i.e., supramolecular assemblies comprised of β-strands. Therefore,measuring the formation of this β-strand secondary structure directly orindirectly via measurement of Arf-Dm2 binding provides the uniqueability to select specific compounds based on their capacity to eithermimic the effect of Arf on Dm2 , or alternatively to interfere with theArf-Hdm2 binding complex and associated β-strand formation. As detailedbelow, such measurements can be performed with NMR spectroscopy,circular dichroism spectropolarimetry, Fourier Transform Infra-Redspectroscopy (FT-IR), fluorescence spectroscopy, or surface plasmonresonance. In addition, determining the size of the resultingprotein-protein, or peptide-peptide complex, can also be used to selectsuch specific compounds. In this case, any size distinguishingmethodology such as gel filtration chromatography, gel electrophoresis,ultra centrifugation, dynamic light scattering etc. may be used.

[0094] Thus, as detailed below, gel filtration chromatographydemonstrated that mArfN37 and Hdm2 210-304 elute together in the voidvolume, whereas the uncomplexed peptides elute at times consistent withmonodisperse, conformationally extended polypeptides. Similarly, NMRresonances for ¹⁵N-mArfN37 or ¹⁵N-Hdm2 210-304 (and ¹⁵N-Hdm2 210-275)are broadened beyond detection when an unlabeled form of the appropriatebinding partner is added to the solution. Furthermore, the NMR spectraare consistent with slow exchange between the free and bound states. Inaddition, at mArfN37:Hdm2 210-304 molar ratios that produce maximalβ-strand secondary structure based on ellipticity at 200 nm using CDresonances cannot be observed for the isotope-labeled component ofArf/Hdm2 mixtures.

[0095] Dm2 or Dm2 peptides comprising one or more specific interactingdomains, such as Hdm2 210-275 and 210-304, exemplified below, can belabeled with ¹⁵N. NMR spectra can be performed and ¹H-¹⁵N steady-state{¹H}-¹⁵N nuclear Overhauser effect (NOE) values can be determined as theratio of peak intensities in 2D ¹H-¹⁵N correlation spectra with andwithout ¹H saturation. Alternatively, or in addition, a circulardichroism spectropolarimeter, as exemplified below, can be used tomonitor the structural changes. Such measurements can be performed inthe presence and absence of test compounds to determine the effect ofthe compound on the formation of β-strand secondary structure in the H1and H2 domains, for example. In one embodiment, the assay is performedin the absence of Arf and Arf fragments, and a compound is selectedwhich can stimulate the formation of β-strand secondary structure of Dm2(fragment thereof) and/or the formation of supramolecular assemblies. Inanother embodiment, the assay is performed in the presence of Arf and/oran Arf fragment, and a compound is selected that can enhance the rate offormation of β-strand secondary structure of Dm2 (or fragment thereof)and/or the formation of supramolecular assemblies. In still anotherembodiment, a compound is selected that interferes with and/or inhibitsthe rate of formation of β-strand secondary structure of Dm2 (fragmentthereof) and/or the formation of supramolecular assemblies in thepresence of Arf and/or an Arf fragment.

[0096] The measurements outlined above can be preceded or alternativelyfollowed by other determinations. Thus, compounds can be initiallyselected for their ability to bind specific Dm2 peptides (such as thosecomprising H1 and H2 as defined in the Example below) or Arf peptides(such as those comprising the amino acid sequence of SEQ ID NO:13).Alternatively, compounds can be selected for their ability to interferewith the binding of Arf with Dm2 (using either the full-length proteinsor fragments, including peptides that comprise the Arf and Dm2 bindingdomains as defined in the Example below). As exemplified below, suchbinding studies can be performed using Surface Plasmon Resonance (SPR).

[0097] Thus, initial screens can be performed in a high throughputformat using any of a large number of methodologies. One such methodemploys a solid support that comprises multiple compartments (e.g.,wells). Currently a solid support comprising between 96 and 1516compartments is relatively standard in drug assays. Each compartment caninclude a separate reaction mixture. In a particular embodiment, aselected target molecule (e.g., an Arf peptide) can be introduced intothe compartments either in solution or on a solid support such as a chip[see U.S. Pat. No. 5,874,219, Issued Feb. 23, 1999, the contents ofwhich are hereby incorporated by reference in their entireties]. Theremaining components of the reaction mixture can be added to thecompartment and a compound can then be added to determine if thecompound binds to the peptide, using radioactive compounds for exampleand a wash step.

[0098] If a chip is employed, a chip reader is generally used to measurethe reaction. Accordingly, the compartments in which the detectablesignal appears can be readily identified. The interaction betweenreactants can be characterized in a number of ways including in terms ofkinetics and/or thermodynamics.

[0099] Assays on biological arrays generally include carrying out theparticular binding reaction under selected conditions, optionallywashing the compartment to remove unreacted molecules, and analyzing thebiological array for evidence of binding between the reactants. Sincethe process can involve multiple steps, it is preferred that such stepsbe automated so as to allow multiple assays to be performedconcurrently. Accordingly high throughput analysis can employ automatedfluid handling systems for concurrently performing the reaction steps ineach of the compartments. Fluid handling allows uniform treatment ofsamples in the compartments. Microtiter robotic and fluid-handlingdevices are commercially available, including from Tecan AG.

[0100] A fluid handling device can be used to manipulate the reactionconditions in any given compartment by, for example, (i) adding orremoving fluid from the compartments, including for manipulating theconcentration of the reactants; (ii) maintaining and/or manipulating thetemperature of the liquid in the compartment; (iii) altering the ionicstrength of the reaction mixture; and (iv) agitating the compartments toensure proper mixing. A reader can then be used to measure the reactionand a computer with an appropriate program can further analyze theresults from the reaction [see U.S. Pat. No. 5,874,219, Issued Feb. 23,1999, the contents of which are hereby incorporated by reference intheir entireties]. Data analysis can include removing “outliers” (datadeviating from a predetermined statistical distribution), andcalculating the relative reaction activity of each compartment. In aparticular embodiment, the resulting data are displayed as an image withcolor in each region varying according to the amount of detectablebinding measured. A solid support can be introduced into a holder in thefluid-handling device.

[0101] Preferably the fluid-handling device is a robotic device that isprogrammed to: (i) set appropriate reaction conditions, such astemperature, and volumes; (ii) to add specific reactants to thecompartments; (iii) incubate the binding partners for an appropriatetime; (iv) remove unreacted reactants; (v) wash the compartments; (vi)add reactants/test compounds as appropriate to the compartments; and(viii) allow the detection of the reaction.

[0102] As part of the binding studies performed herein, it is oftendesiresable to label one or more of the reagents. Suitable labelsinclude enzymes as discussed above, fluorophores (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE), Texas red (TR) rhodamine,free or chelated lanthanide series salts, especially Eu³⁺, to name a fewfluorophores), chromophores, radioisotopes, chelating agents, dyes,colloidal gold, latex particles, ligands (e.g., biotin), andchemiluminescent agents. In the instance where a radioactive label, suchas the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, 90Y,¹²⁵I, ¹³¹I, and ¹⁸⁶Re are used, known currently available countingprocedures may be utilized. In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedtechniques known in the art including ultraviolet, visible, andinfra-red spectroscopy, circular dichroism, magnetic circular dichroism,fluorescence (including measuring changes in fluorescent lifetimes andfluorescent anisotropy), bioluminescence, luminescence, phosphorescence,mass spectrometry, NMR, ESR, amperometric or gasometric techniques.

[0103] Direct labels are one example of labels that can be usedaccording to the present invention. A direct label has been defined asan entity which in its natural state is readily visible, either to thenaked eye or with the aid of an optical filter and/or appliedstimulation, e.g. ultraviolet light to promote fluorescence. Examples ofcolored labels that can be used according to the present inventioninclude metallic sol particles, for example, gold sol particles such asthose described by Leuvering (U.S. Pat. No. 4,313,734); dye solparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as described by May,Supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated inliposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017).Other direct labels include a radionucleotide, a fluorescent moiety or aluminescent moiety. In addition to these direct labeling devices,indirect labels comprising enzymes can also be used according to thepresent invention. Various types of enzyme linked immunoassays are wellknown in the art, for example, alkaline phosphatase and horseradishperoxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactatedehydrogenase, and urease. These and others have been discussed indetail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods inEnzymology, 70:419-439 (1980) and in U.S. Pat. No. 4,857,453. Theprotein/peptides of the present invention can be modified to contain amarker protein such as luciferase or green fluorescent protein asdescribed in U.S. Pat. No. 5,625,048 filed Apr. 29, 1997, WO 97/26333,published Jul. 24, 1997 and WO 99/64592, published Dec. 16, 1999, all ofwhich are hereby incorporated by reference in their entireties. Suitablemarker enzymes include, but are not limited to, alkaline phosphatase andhorseradish peroxidase. Other labels for use in the invention includemagnetic beads or magnetic resonance imaging labels.

[0104] In another embodiment, a phosphorylation site can be created onan antibody of the invention for labeling with ³²P, e.g., as describedin European Patent No. 0372707 (application No. 89311108.8) by SidneyPestka, or U.S. Pat. No. 5,459,240, issued Oct. 17, 1995 to Foxwell etal.

[0105] Polypeptides, and peptides, including antibodies, can be labeledby metabolic labeling. Metabolic labeling occurs during in vitroincubation of the cells that express the protein in the presence ofculture medium supplemented with a metabolic label, such as[³⁵S]-methionine or [³²P]-orthophosphate. In addition to metabolic (orbiosynthetic) labeling with [³⁵S]-methionine, the invention furthercontemplates labeling with [¹⁵N]-amino acids, [¹⁴C]-amino acids and[³H]-amino acids (with the tritium substituted at non-labile positions).

[0106] Once a lead compound is identified it can be tested for itsability to affect one or more of the activities attributed to Arf,including to induce β-strand assembly of Dm2 (e.g., Hdm2 or Mdm2) or afragment thereof. For example, its ability to bind Mdm2, to inhibitMdm2-dependent nucleo-cytoplasmic shuttling of p53, to inhibit the E3ubiquitin ligase activity of Mdm2 toward p53 in vitro and/or its abilityto sequester Mdm2 in the nucleolus can be determined. Such effects canbe measured in Arf^(−/−) cells such as NIH 3T3 cells in which Mdm2 ispresent, for example. Alternatively, microinjection and live cellimaging, as exemplified below can be used to determine whether Hdm2, forexample, is sequestered within nucleoli by a particular compound.

[0107] As detailed below, Hdm2 deletion constructs tagged with afluorescent label (Texas Red™) were microinjected into the nucleus ofNIH 3T3 cells that lack the gene for Arf. Nuclear microinjection wasused because the Hdm2 constructs containing the central Arf-bindingdomain lack the nuclear localization signal found between residues181-186.

[0108] Thus labeled Hdm2 constructs can be individually injected intocells in the absence or presence of the compound and the localization ofthe labeled Hdm2 in the nucleoplasm and nucleoli can be determined. Ifthe labeled Hdm2 is sequestered within nucleoli in the presence of thecompound relative to in its absence, the compound is identified as anArf mimic. Analogously, if the compound interferes with Hdm2 beingsequestered in the nucleoli, when Arf is present, the compound isidentified as an inhibitor of the Arf-Hdm2 interaction. Next, a leadcompound can be tested in animal models to determine its effect ontumors for example, and then finally, tested in humans.

[0109] Furthermore, the effect of a lead compound on cell division canbe determined by monitoring the incorporation of BrdU into chromosomalDNA in NIH 3T3 cells, as previously described [DiGiammarino, et al.,Biochemistry 40:2379-2386 (2001), the contents of which are herebyincorporated by reference in their entireties]. NIH 3T3 cells, forexample, can be cultured in the presence or absence of a lead compoundand, after a set time interval, such as 8 hours, the amount of BrdUincorporated into chromosomal DNA can be determined usingimmunofluorescence microscopy. A lead compound is then selected(identified) when the amount of BrdU incorporated in the presence of thecompound decreases relative to the amount incorporated in the absence ofthe compound.

Antibodies to the Arf and DM2 peptides of the Present Invention

[0110] The Arf and Dm2 polypeptides and peptides of the presentinvention, as produced by a recombinant source or through chemicalsynthesis, or isolated from natural sources, or from a digestion of arecombinant/natural polypeptide, and derivatives or analogs thereof,including fusion proteins, may be used as an immunogen to generateantibodies that recognize the corresponding peptide. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimericincluding humanized chimeric, single chain, Fab fragments, and a Fabexpression library. Polyclonal antibodies have greater likelihood ofcross reactivity.

[0111] Thus the present invention provides compositions and uses ofantibodies that are immunoreactive with the Arf and Dm2 peptides of thepresent invention. Such antibodies “bind specifically” to such peptides,meaning that they bind via antigen-binding sites of the antibody ascompared to non-specific binding interactions. The terms “antibody” and“antibodies” are used herein in their broadest sense, including but notlimited to intact monoclonal and polyclonal antibodies as well asfragments such as Fv, Fab, and F(ab′)2 fragments, single-chainantibodies such as scFv, and various chain combinations. In someembodiments, the antibodies of the present invention are humanizedantibodies or human antibodies. The antibodies may be prepared using avariety of well-known methods including but not limited to immunizationof animals having native or transgenic immune repertoires, phagedisplay, hybridoma and recombinant cell culture, and transgenic plantand animal bioreactors.

[0112] Both polyclonal and monoclonal antibodies may be prepared byconventional techniques. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

[0113] Various procedures known in the art may be used for theproduction of polyclonal antibodies to the Arf and Dm2 peptides of thepresent invention or derivatives or analogs thereof. For the productionof antibody, various host animals can be immunized by injection withsuch a peptide, or a derivative (e.g., or fusion protein) thereof,including but not limited to rabbits, mice, rats, sheep, goats, etc. Inone embodiment, the peptide can be conjugated to an immunogenic carrier,e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

[0114] For preparation of monoclonal antibodies directed toward the Arfand Dm2 peptides of the present invention, or analogs, or derivativesthereof, any technique that provides for the production of antibodymolecules by continuous cell lines in culture may be used. These includebut are not limited to the hybridoma technique originally developed byKohler and Milstein [Nature, 256:495-497 (1975)], as well as the triomatechnique, the human B-cell hybridoma technique [Kozbor et al.,Immunology Today, 4:72 (1983); Cote et al., Proc. Natl. Acad. Sci.U.S.A., 80:2026-2030 (1983)], and the EBV-hybridoma technique to producehuman monoclonal antibodies [Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology [PCT/US90/02545].

[0115] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., “humanized” versions of antibodies originallyproduced in mice or other non-human species. Such humanized antibodiesmay be prepared by known techniques and offer the advantage of reducedimmunogenicity when the antibodies are administered to humans. In fact,according to the invention, techniques developed for the production of“chimeric antibodies” [Morrison et al., J. Bacteriol., 159:870 (1984);Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature,314:452-454 (1985)] by splicing the genes from a mouse antibody moleculespecific for an Arf and/or Dm2 peptide of the present invention togetherwith genes from a human antibody molecule of appropriate biologicalactivity can be used. Antibodies such as these are within the scope ofthis invention.

[0116] Thus, a humanized antibody is an engineered antibody thattypically comprises the variable region of a non-human (e.g., murine)antibody, or at least complementarity determining regions (CDRs)thereof, and the remaining immunoglobulin portions derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described Riechmann etal., [Nature 332:323, (1988)]; Liu et al., [Proc.Nat.Acad.Sci. 84:3439(1987)]; Larrick et al., [Bio/Technology 7:934, (1989)]; and Winter andHarris, [TIBS 14:139, (May, 1993)]. Such human or humanized chimericantibodies are preferred for use in therapy of human diseases ordisorders (described infra), since the human or humanized antibodies aremuch less likely than xenogenic antibodies to induce an immune response,in particular an allergic response, themselves.

[0117] Therefore, procedures that have been developed for generatinghuman antibodies in non-human animals may be employed in producingantibodies of the present invention. The antibodies may be partiallyhuman or preferably completely human. For example, transgenic mice intowhich genetic material encoding one or more human immunoglobulin chainshas been introduced may be employed. Such mice may be geneticallyaltered in a variety of ways. The genetic manipulation may result inhuman immunoglobulin polypeptide chains replacing endogenousimmunoglobulin chains in at least some, and preferably virtually all,antibodies produced by the animal upon immunization. Mice in which oneor more endogenous immunoglobulin genes have been inactivated by variousmeans have been prepared. Human immunoglobulin genes have beenintroduced into the mice to replace the inactivated mouse genes.Antibodies produced in the animals incorporate human immunoglobulinpolypeptide chains encoded by the human genetic material introduced intothe animal. Examples of techniques for the production and use of suchtransgenic animals to make antibodies (which are sometimes called“transgenic antibodies”) are described in U.S. Pat. Nos. 5,814,318,5,569,825, and 5,545,806, which are incorporated by reference herein.

[0118] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also provided by the presentinvention. Such hybridomas may be produced and identified byconventional techniques. One method for producing such a hybridoma cellline comprises immunizing an animal with a polypeptide, harvestingspleen cells from the immunized animal, fusing said spleen cells to amyeloma cell line, thereby generating hybridoma cells, and identifying ahybridoma cell line that produces a monoclonal antibody that binds thepolypeptide. The monoclonal antibodies produced by hybridomas may berecovered by conventional techniques.

[0119] According to the invention, techniques described for theproduction of single chain antibodies [U.S. Pat. Nos. 5,476,786 and5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to producee.g., Arf peptide-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries [Huse et al., Science,246:1275-1281 (1989)] to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for an Arfpeptide, or its derivatives, or analogs.

[0120] Antibody fragments which contain the idiotype of the antibodymolecule can be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab′)₂ fragment which canbe produced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

[0121] In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art, e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), Western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. Antibody binding can be detected by detecting a label onthe primary antibody, or by detecting binding of a secondary antibody orreagent to the primary antibody. In an alternative embodiment, thesecondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of Arf, one may assay generated hybridomas for aproduct which binds to the Arf fragment containing such epitope andchoose those which do not cross-react with the full-length Arf protein.

[0122] In a specific embodiment, antibodies that agonize or antagonizethe binding of Arf to Hdm2 can be generated. Such antibodies can betested using the assays described infra. In addition an antibody thatmimics the effect of Arf on Hdm2 can also be assayed for using theassays disclosed herein.

Administration of the Therapeutic Compositions of the Present Invention

[0123] According to the present invention, the component or componentsof a therapeutic composition of the invention may be introducedtopically, parenterally, transmucosally, e.g., orally, nasally, orrectally, or transdermally. When the administration is parenteral, itmay be via intravenous injection, and also including, but is not limitedto, intra-arteriole, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration.

[0124] In a particular embodiment, the therapeutic compound can bedelivered in a vesicle, in particular a liposome [see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss:New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.].

[0125] In yet another embodiment, the therapeutic compound can bedelivered in a controlled release system. For example, a small organicmolecule as described above, may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used[see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)]. In another embodiment, polymeric materials can be used[see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)]. In yet another embodiment, a controlled release system can beplaced in proximity of a therapeutic target, thus requiring only afraction of the systemic dose [see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)].

[0126] Other controlled release systems are discussed in the review byLanger [Science 249:1527-1533 (1990)].

[0127] Thus, a therapeutic composition of the present invention can bedelivered by intravenous, intraarterial, intraperitoneal, intramuscular,or subcutaneous routes of administration. Alternatively, the therapeuticcomposition, properly formulated, can be administered by nasal or oraladministration. A constant supply of the therapeutic composition can beensured by providing a therapeutically effective dose (i.e., a doseeffective to induce metabolic changes in a subject) at the necessaryintervals, e.g., daily, every 12 hours, etc. These parameters willdepend on the severity of the condition being treated, other actions,such as diet modification, that are implemented, the weight, age, andsex of the subject, and other criteria, which can be readily determinedaccording to standard good medical practice by those of skill in theart.

[0128] A subject in whom administration of the therapeutic compositionis an effective therapeutic regiment for cancer treatment for example,is preferably a human, but can be any primate, other mammals or evenavians suffering from cancer, including domestic animals such as dogsand cats, laboratory animals such as rats, rabbits and mice, livestock,such as cattle (including cows), pigs, horses, and goats, and animalsmaintained in a zoo such as elephants, lions, zebras, and gorillas.Thus, as can be readily appreciated by one of ordinary skill in the art,the methods and pharmaceutical compositions of the present invention areparticularly suited to administration to a number of animal subjects,but particularly humans.

Kits

[0129] The materials for use in this aspect of the invention are ideallysuited for the preparation of a kit. Specifically, the inventionprovides a compartmentalized kit to receive in close confinement, one ormore containers which comprise one or more of the peptides of thepresent invention; and optionally one or more other containerscomprising reagents, such as additional buffers etc.

[0130] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow one to efficiently transfer reagents fromone compartment to another compartment such that the samples andreagents are not cross-contaminated and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the peptides used in the assay,and containers which contain additional reagents such as specificbuffers with defined ionic strengths.

[0131] The present invention may be better understood by reference tothe following non-limiting Example, which is provided as exemplary ofthe invention. This example is presented in order to more fullyillustrate the preferred embodiments of the invention.

[0132] It should in no way be construed, however, as limiting the broadscope of the invention.

EXAMPLE Defining the Molecular Basis of Arf And Hdm2 InteractionsIntroduction

[0133] Understanding the interaction of Arf and Hdm2 has recently becomea central issue in cancer biology. In response to hyperproliferativesignals, p14^(Arf) stabilizes p53 by binding to Hdm2 and inhibits theubiquitination and subsequent proteosome-dependent degradation of p53.The medical importance of the Arf-Hdm2-p53 regulatory system ishighlighted by the finding that either p53 or p14^(Arf) are lost ormodified in virtually all human cancers.

[0134] Human and mouse Arf are highly basic proteins (˜20% Arg residues)of 132 and 169 residues, respectively, that localize to nucleoli. Theextreme N-terminal segments of the two proteins are very similar (17/29identity; 21/29 similarity) and contain a repeated motif of 8 or 9residues that comprises hydrophobic residues flanked by Arg residues[DiGiammarino et al., Biochem., 40:2379-2386 (2001)]. Exon 1β of thehuman and mouse p16^(Ink4a)/Arf locus uniquely encodes the first 62 and63 amino acids of human and mouse Arf, respectively, while exon 2encodes the C-terminal domains. An alternative reading frame within exon2 also encodes the central segment of p16^(Ink4a) [Quelle et al., Cell,83:993-1000 (1995)]. Importantly, peptides containing highly conservedN-terminal residues of human or mouse Arf have been shown to possessbiological activity [Midgley et al., Oncogene, 19:2312-23 (2000); Weberet al., Mol. Cell Biol., 20:2517-2528 (2000); DiGiammarino et al.,Biochem., 40:2379-2386 (2001)]. For example, a peptide containing theN-terminal 37 amino acids of mouse Arf (termed mArfN37) localizes tonucleoli, binds and sequesters Hdm2 within nucleoli, and activates p53leading to cell cycle arrest [Weber et al., Mol. Cell Biol.,20:2517-2528 (2000); DiGiammarino et al., Biochem., 40:2379-2386(2001)]. Additionally, a 20 amino acid peptide from the human ArfN-terminus inhibits Mdm2-dependent ubiquitination of p53 and activatesp53 in vivo [Midgley et al., Oncogene, 19:2312-23 (2000)]. Further studyof the Arf N-terminus has shown that the two repeated motifs withinmArfN37 bind individually to Hdm2 and are both required for normal Arffunction [Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)]. Twosegments of p14^(Arf) also are reported to mediate interactions withHdm2 but one of these is found in a different region of the polypeptide;these are residues 1-14 and 82-101 [Weber et al., Mol. Cell Biol.,20:2517-2528 (2000); Zhang et al., Mol. Cell, 3:579-91 (1999)].Nucleolar localization of mouse and human Arf is specified by the aminoacid sequence RRPR (SEQ ID NO:14) (the nucleolar localization signal,NoLS); the NoLS in p19^(Arf) is found between residues 31-34 of SEQ IDNO:2 and in p14^(Arf) between residues 87-90 of SEQ ID NO:4.Interestingly, when Arf binds Mdm2 (or Hdm2), the Arf NoLS is masked andnucleolar colocalization of the Arf/Mdm2 complex relies on the exposureof a cryptic NoLS within the RING domain of Mdm2 [Lohrum et al., Nat.Cell Biol., 2:179-81 (2000); Weber et al., Mol. Cell Biol., 20:2517-2528(2000)]. Despite the wealth of information on how Arf functions incells, detailed information on the Hdm2-bound state of mArfN37, or themechanism of Hdm2 binding or nucleolar localization is completelylacking.

[0135] Mdm2 is a multifunctional protein that is reported to interactwith p53 [Wu et al., Genes Dev., 7:1126-1132 (1993)], CBP/p300 [Grossmanet al., Mol Cell, 2:405-15 (1998)], E2F1 [Martin et al., Nature,375:691-4 (1995); O° C.onnor et al., Embo J, 14:6184-92 (1995)], Rb[Xiao et al, Nature, 375:694-8 (1995)], L5 [Marechal et al., Mol CellBiol, 14:7414-20 (1994)], TBP [Leveillard et al., Mech Dev, 74:189-93(1998)] and Arf [Weber et al., Nat. Cell Biol., 1:20-26 (1999); Weber etal., Mol. Cell Biol., 20:2517-2528 (2000); DiGiammarino et al.,Biochem., 40:2379-2386 (2001)]. The human (Hdm2) and mouse (Mdm2)orthologs are 72% identical and can functionally substitute for oneanother. The domains responsible for some of the above interactions arewell characterized. For example, the N-terminal domain of ˜100 aminoacids adopts a globular, helical structure and binds a small peptidewithin the p53 N-terminus; this interaction inhibits the transcriptionalactivation function of p53 [Kussie et al., Science, 274:948-953 (1996);Oliner et al., Nature, 362:857-860 (1993); Momand et al., Cell,69:1237-1245 (1992)]. Two zinc-binding motifs have been identified inMdm2, a C₄ zinc finger motif (amino acid residues 305-325 of SEQ IDNO:6) and a C₃HC₄ RING domain (amino aid residues 438-478 of SEQ IDNO:6) [Boddy et al., Trends Biochem Sci, 19:198-9 (1994)]. The latterdomain has been shown to mediate ubiquitin ligase activity toward p53 invitro [Honda et al., FEBS Lett, 420:25-7 (1997); Honda et al., Embo J,18:22-7 (1999)] and to bind RNA [Elenbaas et al., Mol. Med., 2:439-51(1996); Lai et al., Biochem., 37:17005-15 (1998)]. Further, the RINGdomain has been shown to bind zinc ions and to exhibit globular butdisordered structure [Lai et al., Biochem., 37:17005-15 (1998)]. The C₄zinc finger motif has not been functionally characterized. TheArf-binding domain of Hdm2 has been mapped to amino acids 210-304 of SEQID NO:8 (termed Hdm2 210-304) [Weber et al., Mol. Cell Biol.,20:2517-2528 (2000)]. The L5 binding domain also maps to this region[Marechal et al., Mol Cell Biol, 14:7414-20 (1994)]. Between humans andmice, this segment is 92% similar and, in striking contrast to Arg-richArf, is highly acidic (for Hdm2, 32% Asp/Glu, predicted pI ˜3.2; forMdm2, 33% Asp/Glu; predicted pI ˜3.5). This central segment has beenshown to bind N-terminal fragments of mouse Arf, including 1-37, 1-14and 26-37 [Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)].Furthermore, a peptide composed of the first 20 amino acids of humanp14^(Arf) has been shown to bind the central, acidic segment of Hdm2 andto inhibit Hdm2-dependent ubiquitination of p53 in vitro [Midgley etal., Oncogene, 19:2312-23 (2000)]. The interaction motif within Hdm2 hasbeen mapped to residues 212-244 [Midgley et al., Oncogene, 19:2312-23(2000)]. The studies summarized above contribute significantly to theunderstanding of the cellular functions of Arf and Hdm2 but providelittle insight into the physical and structural basis for thesefunctions, such as the binding of Arf to Hdm2, nucleolar colocalization,inhibition of Hdm2-dependent nucleo-cytoplasmic shuttling of p53, and E3ubiquitin ligase activity toward p53.

Materials and Methods

[0136] Hdm2 and p19^(Arf) Protein Purification:

[0137] Fragments of Hdm2 corresponding to residues 210-275 and 210-304of SEQ ID NO:8 (termed Hdm2 210-275 and Hdm2 210-304) were subclonedinto the expression plasmid pET28a (Novagen) using standard methods;pET28a allows expression of polypeptides with a thrombin cleavablepoly-His affinity purification tag. Following protein expression in E.coli BL21 (DE3) (Novagen, Inc.), bacterial cells were harvested bycentrifugation followed by resuspension in 20 mM Tris-HCl (pH 8.0) 500mM NaCl at 4° C., and lysed by sonication. Urea was added to a finalconcentration of 6 M to the soluble fraction after centrifugation(20,000 g, 20 min.). Soluble, His-tagged proteins were purified usingNi²⁺-affinity chromatography (Chelating-Sepharose, Amersham PharmaciaBiotech, Inc.) in the presence of 6 M urea using otherwise standardprocedures (Novagen, Inc.). An N-terminal fragment of mouse p19^(Arf)corresponding to residues 1-37 (mArfN37) was purified in a similarmanner, as previously reported [DiGiammarino et al., Biochem.,40:2379-2386 (2001)]. His tag-cleaved mArfN37 was further purified usingC₄ reverse-phase high-performance liquid chromatography (HPLC) (C₄column; Vydac, Inc.) using a 0.1% trifluoroacetic acid(TFA)/acetonitrile buffer system. Lyophilized proteins were directlydissolved in the appropriate buffer for each experiment, as describedbelow. Fractions containing Hdm2 proteins were dialyzed into 20 mMTris-HCl (pH 8.0), 500 mM NaCl and treated with thrombin (Novagen) 1U/mg protein at room temperature for 16 hours to cleave the His tag.Constructs were further purified using anion exchange chromatography (QSEPHAROSE, Amersham Pharmacia Biotech, Inc.) using 20 mM Tris-HClbuffer, pH 7.0 with elution using a 50 mM—1.0 M NaCl gradient over 0.05liters.

[0138] Peptide Synthesis.

[0139] Peptides were synthesized using standard methods by an AdvancedChemtech 396 synthesizer. The peptide amides were synthesized onHMP-amide resin (Applied Biosystems, Inc.) and the FMOC-amino acids werecoupled using HOBt/HBTU chemistries. N-terminal acetylation wasperformed using acetic anhydride and HOBt. Peptides were cleaved fromthe resin in 91% TFA containing 2% phenol, 2% ethanedithiol and 5%thioanisole. The peptides were precipitated and washed twice withdiethyl ether. Peptide concentrations for circular dichroism (CD) andsurface plasmon resonance (SPR) were determined using quantitative aminoacid analysis.

[0140] Surface Plasmon Resonance:

[0141] Binding studies were performed using a Biacore 3000 surfaceplasmon resonance (SPR) instrument (Biacore, Inc.). A Tetra-His™Antibody (Qiagen, Inc.; Cat. #34670) was covalently attached to acarboxymethylated gold surface (C-1 chip; Biacore, Inc.). Thecarboxymethyl groups on the surface were activated withN-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) and the antibody was attached at pH 7.4 in 20mM sodium phosphate buffer, 150 mM NaCl (PBS). Reactive sites remainingon the surface were blocked by reaction with ethanolamine. TheHis-tagged ligand was attached to the antibody by injecting a 5-10 μg/mlsolution of the ligand in 10 mM HEPES, 150 mM NaCl, pH 7.4 (HBS-Nbuffer, Biacore) at a flow rate of 10 μL/min through the flow cell. Areference cell was prepared similarly except that no His-tagged ligandwas added. Binding was measured by flowing the non-His-tagged analytethrough the reference and ligand-containing flow cells in sequence.Prior to injection of ligands, the chip surfaces were equilibrated inHBS-N buffer. Changes in the SPR response due to solvent differences andthe injection process were monitored by injection of HBS-N buffer alone.Regeneration of the chip surface to remove bound analyte and ligand wasaccomplished by two 100 μL injections of 10 mM glycine, pH 2.0 throughboth flow cells. Data reported is the difference in SPR signal betweenthe flow cell containing analyte and the reference cell. Duplicateinjections were made and the SPR response values reported are theaverage of these two injections.

[0142] NMR Spectroscopy:

[0143] Uniformly ¹⁵N-labeled Hdm2 210-275 and 210-304 were preparedusing standard procedures [Kriwacki et al., Proc. Natl. Acad. Sci. USA,93:11504-11509 (1996)]. Samples were concentrated by ultra-filtrationusing a 3000 Da cutoff filter (Millipore, Corp., Centricon 3) to 1-2 MMin 10 mM potassium phosphate buffer, pH 6.0, 5% D₂O (vol:vol) and placedin 5 mm micro-cell NMR tubes (Shegimi, Inc.). All NMR spectra wereacquired using a 600 MHz Varian Inova NMR spectrometer (VarianAssociates, Inc.) fitted with a 5 mm triple resonance probe equippedwith x, y, z axis pulsed magnetic field gradients. 2D ¹H-¹⁵N HSQCspectra [Muller, J. Amer. Chem. Soc., 101:4481-4484 (1979); Bodenhausenand Ruben, D. J. Chem. Phys. Lett., 69:185-189 (1980)] were acquiredusing standard procedures provided in the Varian Protein Pack pulsesequence library. ¹H-¹⁵N steady-state {¹H}-¹⁵N nuclear Overhauser effect(NOE) values were determined as the ratio of peak intensities in 2D¹H-¹⁵N correlation spectra with and without ¹H saturation [Farrow etal., Biochem., 33:5984-6003 (1994)].

[0144] Circular Dichroism:

[0145] Spectra were recorded at 25° C. using an Aviv Model 62A DScircular dichroism spectropolarimeter (Aviv Instruments, Inc.) equippedwith a thermoelectric temperature control unit. All samples wereprepared in 10 mM Tris-HCl, pH 7.0 at 0.02-0.2 mM as determined by aminoacid analysis. Spectra were recorded using 1 mm quartz cuveftes andreported spectra are the average of 10 scans over the range 195-260 nMin 1 nM steps. Melting experiments were recorded in 10 mm quartzcuvettes with active stirring. The signal at 216 nm (β strand minimum)was used to monitor structural changes and the signal was averaged over15 sec. The sample temperature was increased in 1° C. steps.

[0146] Fluorescent Labeling:

[0147] Proteins for fluorescence microimaging studies were covalentlymodified using an amine-reactive, sulfosuccinimidyl ester of Texas Red™(Molecular Probes, Inc., Cat. # C-1171). In brief, purified Hdm2-derivedproteins were dialyzed into 100 mM sodium bicarbonate buffer, pH 8.3 at2-4 mg/ml. The Texas Red™ dye was dissolved in dimethylsulfoxide at 10mg/ml. Protein and dye 3:1 (vol:vol) were reacted for 1 hour at roomtemperature followed by purification using size-exclusion chromatography(NAP 5 G-25 columns, Amersham Pharmacia Biotech, Inc.). Labeled proteinbands migrated more slowly than unlabeled protein in SDS-polyacrylamidegels consistent with the covalent attachment of dye.

[0148] Cell Culture and Microinjection:

[0149] NIH 3T3 Arf^(−/−) cells passage 10-15 were cultured in Dulbecco'smodified Eagle's medium (Dulbecco's modified Eagle's medium (DMEM);Gibco-Invitrogen, Inc.), 10% fetal bovine serum (FBS) at 37° C., 7% CO₂.24 hours prior to transfection, cells were plated on 35 mm dishes coatedwith poly-D-Lysine (Mattek, Corp.) at a density of 3×10⁴ cells per dish.Cells were transfected with a pcDNA plasmid (Invitrogen) containing theGFP-p19^(ARF) fusion protein using Fugene-6 (Roche MolecularBiochemicals, Inc.) in media according to the protocol supplied by themanufacturer. Sixteen hours post-transfection, the transfection mediawas refreshed with DMEM, 10% FBS and the cells were allowed to recoverfor four hours. GFP-Arf positive cells were then located and co-injectedwith Texas Red-labeled Hdm2 polypeptides (2 mg/ml in 20 mM Tris-HCl, 100mM NaCl, pH 7.0) using a Micromanipulator 5171/Transjector 5246(Eppendorf, AG) in media supplemented with 10 mM HEPES, pH 7.2 to buffercell media during injection and imaging. Representative cells wereimaged over time. An Axiovert 135 TV inverted fluorescence microscopewith an automated stage controller (Carl Zeiss, Inc.) and circulatingwater bath heater (Fisher) was used for both microinjection and imaging.Images were acquired with a Zeiss 40× NA=1.30 oil objective and MicroMaxCCD camera (Princeton Instruments Inc.) operated with MetaMorph Version4.01 imaging software.

Results

[0150] Small Segments of Arf and Hdm2 Participate in Arf/Hdm2Interactions.

[0151] It has been previously shown that a fragment of mouse p19^(Arf)containing the N-terminal 37 amino acids (mArfN37) can (i) bind Hdm2,(ii) cause the relocalization of Hdm2 to nucleoli, and (iii) induce cellcycle arrest in MEFs [see, U.S. patent application Ser. No. 09/480,718,Filed Jan. 7, 2000, the contents of which are hereby incorporated byreference in their entireties; Weber et al., Nat. Cell Biol., 1:20-26(1999)]. Further, a fragment of Hdm2 containing amino acids 140-350 hasbeen show to be capable of binding mArfN37 and to be relocalized tonucleoli in a mArfN37-dependent manner. A smaller fragment, Hdm2210-304, has also been shown to be capable of binding mArfN37 on thebasis of Arf affinity chromatography [Weber et al., Mol. Cell Biol.,20:2517-2528 (2000)]. However, because mArfN37 is a relatively smallpolypeptide, it was determined whether it could bind a correspondinglysmall segment of Hdm2. To test this hypothesis and to monitor Arf/Hdm2binding reactions quantitatively, Hdm2 constructs were prepared spanningamino acids 210-304 of SEQ ID NO:8 and 210-275 of SEQ ID NO:8 and thenit was determined whether they could bind mArfN37 using surface plasmonresonance (SPR). Both Hdrn2 210-304 and 210-275 bind tightly toHis-tagged mArf37 that was immobilized on the SPR biosensor surfaceusing a covalently linked His antibody (FIG. 1). The two Hdm2 fragmentsdid not bind a control surface that lacked His-mArfN37.

[0152] The binding results discussed above were verified (based on an invitro assay using SPR) in a biological setting by monitoring theinteraction of p19^(Arf) and Hdm2 fragments in NIH 3T3 cells that lackthe gene for Arf using fluorescence microscopy. p19^(Arf) was taggedwith GFP (green fluorescent protein) and expressed in cells aftertransfection with an expression plasmid, as previously described [Weberet al., Nat. Cell Biol., 1:20-26 (1999); Weber et al., Mol. Cell Biol.,20:2517-2528 (2000)], while Hdm2 was chemically tagged with thefluorescent dye Texas Red™ and introduced into cells by microinjection.The results show that GFP-p19^(Arf) is localized in nucleoli aftertransfection, whereas, in direct contrast, Hdm2 210-304 is evenlydispersed in the nucleoplasm after microinjection in the absence ofp19^(Arf). When GFP-p19^(Arf) and Hdm2 210-304 are introduced into cellstogether, the two proteins become co-localized within nucleoli. Thesmaller fragment of Hdm2, Hdm2 210-275, exhibits similar localizationproperties in the absence and presence of GFP-p19^(Arf). Together, theSPR and cell localization results show that a ˜100 amino acid centralsegment of Hdm2 interacts with Arf and that fragments containing thissegment can be sequestered within nucleoli in the same manner as shownpreviously for full-length Hdm2 [Weber et al., Nat. Cell Biol., 1:20-26(1999); Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)].

[0153] Structural Properties of mArfN37 and Arf-binding Hdm2 Fragments.

[0154] Knowing that mArfN37 and Hdm2 210-304 (and Hdm2 210-275) interactand that the interactions appear to be biologically relevant, thestructural properties of these domains were investigated. Previous workshowed that mArfN37 is unstructured in aqueous solution and that thepeptide adopts a bi-helical conformation in 30% trifluoroethanol[DiGiammarino et al., Biochem., 40:2379-2386 (2001)]. CD spectra forHdm2 210-304 and 210-275 show that these two Arf-binding polypeptidesare also unstructured. The CD spectrum for mArfN37 is also shown, forreference. A similar conclusion can be reached on the basis of the¹H-¹⁵N 2D correlation spectra for Hdm2 210-304 and 210-275. The observedchemical shift values in both the ¹H and ¹⁵N dimensions cluster nearrandom coil values [Schwarzinger et al., J. Amer. Chem. Soc.,123:2970-2978 (2001)] and are consistent with a general lack ofsecondary and tertiary structure [Kriwacki et al., Proc. Natl. Acad.Sci. USA, 93:11504-11509 (1996)]. Furthermore, heteronuclear {¹H}-¹⁵NNOE values for Hdm2 210-275 are all negative and are consistent with theconclusion that the Arf-binding segment of Hdm2, prior to binding Arf,is conformationally disordered and highly flexible.

[0155] Secondary structure prediction methods were used to gain furtherinsight into the structural properties of the N-terminal segment of Arf,and the Arf-binding segment of Hdm2. Prior analysis of mArfN37 aloneusing a variety of secondary structure prediction algorithms did notyield consistent results [DiGiammarino et al., Biochem., 40:2379-2386(2001)]. However, when the neural net-based Jnet algorithm [Cuff et al.,Proteins, 40:502-11 (2000)] (available through the Jpred site atjura.ebi.ac.uk:8888) was used to analyze the human, mouse, and opossumArf sequences simultaneously, two short β-strands are predicted withinthe N-terminal 37 amino acids (FIG. 2a), between residues 4-12 and20-27, respectively. The Jnet approach, which is based on the principlethat secondary structure is conserved within evolutionarily relatedproteins, has been shown to predict secondary structure with 73% orhigher accuracy. For Arf, both β-strands are predicted with highconfidence (FIG. 2a). Prior structure predictions were probably hamperedby the unusual nature of the mArfN37 sequence, which contains 27% Argresidues (10/37) and has a correspondingly high predicted pI value(12.6).

[0156] In contrast to mArfN37, Hdm2 210-304 is highly acidic (17/95 Aspand 14/95 Glu, 31/95 total, or 33%; the predicted pI is 3.5) and veryhydrophilic due additionally to its high Ser content (17/95).Interestingly, as for the Arf N-terminus, two short segments of β-strandare predicted by Jnet within the Arf-binding domain of Hdm2, betweenresidues 245-253 and 275-282 (FIG. 2b). Many residues within theβ-strands are also predicted to be less than 25% solvent exposed. Theremainder of the polypeptide is predicted to be unstructured and solventexposed. The opposed charge characteristics of mArfN37 and Hdm2 210-304suggest that electrostatic forces play a role in the interactionsbetween the two polypeptides. The prediction of two β-strands in bothmArfN37 and Hdm2 210-304 suggests that β-strand secondary structure isinvolved in Arf/Hdm2 interactions.

[0157] β-strand Secondary Structure Forms when Arf and Hdm2 Interact.

[0158] Interestingly and in accord with the secondary structurepredictions discussed above, a striking transition from randomconformations to β-strand secondary structure is observed when mArfN37and Hdm2 210-304 (and Hdm2 210-275) are mixed (FIG. 3). This structuraltransition is induced when mArfN37 is added to an Hdm2 fragment and whenan Hdm2 fragment is added to mArfN37. Data from gel filtrationchromatography and NMR spectroscopy shows that the mArfN37 and Hdm2210-304 do not form a bimolecular complex involving small numbers ofmolecules but rather form large, extended structures with predominantlyβ-strand secondary structure. First, gel filtration chromatography showsthat, when mixed, mArfN37 and Hdm2 210-304 elute together in the voidvolume. In contrast, the uncomplexed species elute at times consistentwith monodisperse, conformationally extended polypeptides. Second, NMRresonances for ¹⁵N-mArfN37 or ¹⁵N-Hdm2 210-304 (and ¹⁵N-Hdm2 210-275)are broadened beyond detection when an unlabeled form of the appropriatebinding partner is added to the solution. At mArfN37:Hdm2 210-304 (ormArfN37:Hdm2 210-275) molar ratios that produce maximal β-strandsecondary structure based on ellipticity at 200 nm using CD, resonancescannot be observed for the isotope-labeled component of Arf/Hdm2mixtures. Further, the NMR spectra are consistent with slow exchangebetween the free and bound states. These results indicate that β-strandsecondary structure forms when mArfN37 and Hdm2 210-304 (and Hdm2210-275) interact and that this secondary structure exists in thecontext of supramolecular assemblies that can be described as βnetworks.

[0159] Characterization of Arf:Hdm2 Assemblies.

[0160] As discussed above, the addition of mArfN37 to Hdm2 210-304results in the formation of supramolecular assemblies comprised ofβ-strands. Stepwise addition of sub-stoichiometric amounts of mArfN37 toHdm2 210-304 increased β-strand secondary structure content as judged bythe change in ellipticity at 200 nm using CD. Ellipticity at 200 nm wasmonitored because the largest difference in ellipticity between randomand β-strand conformations was observed at this wavelength. The bindingof mArfN37 to Hdm2 210-304, as monitored in this way, is saturable.Similar results were obtained when mArfN37 was mixed with the shorterfragment of Hdm2, Hdm2 210-275. Further, saturable binding is alsoobserved when the Hdm2 fragments were added to an excess of mArfN37.These findings indicate that there are a limited number of binding sitesfor mArfN37 within Hdm2 210-304 and that discreet structures (containingβ-strands) form when mArfN37 and Hdm2 210-304 (or 210-275) are mixed.For mArfN37 added to Hdm2 210-304, the plot of ellipticity versus amountof protein added is linear, indicating that each molecule of mArfN37added binds completely to the Hdm2 fragment. Similar behavior isobserved when mArfN37 binds Hdm2 210-275. These findings suggest thatthe equilibrium dissociation constant (K_(D)) for the interactions isless than the concentrations used (˜5×10⁶ M).

[0161] Formation of mArfN37:Hdm2 210-304 (and mArfN37:Hdm2 210-275)assemblies was observed under a variety of conditions, including pHvalues between 4 and 10, and salt concentrations from 0 to 2 M NaCl.Furthermore, the assemblies were not disrupted by chemical denaturation(4 M urea), treatment with an organic solvent (50% acetonitrile(vol:vol)), or treatment with a mild detergent (10 mM CHAPSO) Theseresults indicate that the mArfN37:Hdm2 210-304 assemblies arethermodynamically stable. This conclusion is further supported by theresults of thermal denaturation experiments using CD. The assemblyformed by adding mArfN37 to an excess of Hdm2 210-304 to a final molarratio of 2:1 (mArfN37:Hdm2 210-304) is stable to heating up to ˜75° C.For example, the CD spectrum for the mArfN37:Hdm2 210-304 assemblyprepared in this way does not change when the sample temperature isincreased from 25° C. to 75° C. The CD spectrum does change above 75°C., and the changes are consistent with unfolding of the supramolecularassemblies. In particular, the intensity of spectral features indicativeof β-strands is reduced and finally completely eliminated at 95° C. WhenCD ellipticity at 216 nm is plotted versus temperature, the shape of thecurve above 75° C. resembles other protein denaturation curves that areknown to involve a cooperative unfolding process. The melting data wasfit with a sigmoidal function to determine the melting temperature(T_(m)), defined as the midpoint of the melting curve. Denaturationexperiments were performed at three different ionic strengths, 0, 150and 300 mM NaCl, and yield T_(m) values of 77, 65 and 52° C.,respectively. Similar T_(m) values were obtained with the assemblyformed by adding mArfN37 to Hdm2 210-275.

[0162] Dual Binding Motifs in Arf and Hdm2 Mediate IntermolecularInteractions.

[0163] The N-terminal domains of human and mouse Arf have similarprimary structure (FIG. 2a) and contain an arginine-rich, repeated motif(FIG. 4) [DiGiammarino et al., Biochem., 40:2379-2386 (2001)]; this istermed the “Arf motif” the first and second repeats are termed “A1” and“A2”, respectively. [In the mouse Arf protein (p19^(Arf)) A1 and A2 havethe amino acid sequences of SEQ ID NO:s 9 and 10 respectively, whereasin the human Arf (p14^(Arf)), A1 and A2 have the amino acid sequences ofSEQ ID NOs:11 and 12 respectively.

[0164] Importantly, mArfN37, which contains both of the Arf motifs inthe mouse Arf sequence, has been shown to possess biological propertiescomparable to full-length mouse Arf. These properties include nucleolarlocalization, the ability to sequester Mdm2 and Hdm2 in nucleoli and theability to cause cell cycle arrest [Weber et al., Mol. Cell Biol.,20:2517-2528 (2000); DiGiammarino et al., Biochem., 40:2379-2386(2001)]. Furthermore, a construct containing residues 1-20 of human Arfhas been shown to activate p53 in cellular assays [Midgley et al.,Oncogene, 19:2312-23 (2000)]. The existence of two structural motifswithin mArfN37 was suggested on the basis of the solution structure ofmArfN37 determined in the presence of trifluoroethanol (TFE) using NMRspectroscopy [DiGiammarino et al., Biochem., 40:2379-2386 (2001)]. InTFE, mArfN37 is bi-helical, with the two Arf motifs contained by helicesthat are 12 amino acids in length. Based on the observation thatβ-strands form when mArfN37 and Hdm2 210-304 interact, and theprediction of β-strands within the interacting segments, the bi-helicalconformation of mArfN37 in TFE is probably not relevant to theHdm2-bound conformation. However, the observation of similar structurefor the mouse A1 and A2 segments in mArfN37 did suggest that the two Arfmotifs may function in Hdm2 binding in a structurally similar andmechanistically coordinated manner. With these ideas in mind, the rolethe two Arf motifs play in Hdm2 binding was investigated by monitoringthe binding of short peptides derived from human and mouse Arf to Hdm2fragments using surface plasmon resonance and CD.

[0165] Two libraries of peptides 15 amino acids in length weresynthesized based on the sequence of exon 1β of mouse p19^(Arf) and theentire sequence of human p14^(Arf). The sequence for the first peptidein each library corresponded to the first 15 amino acids of the proteinsequences. The N-terminus for the second peptide was shifted forward 5residues to position 6 and spanned residues 6-20, and each subsequentpeptide had an additional 5-residue forward shift of the N-terminus.This approach yielded two libraries of overlapping peptides spanningmouse p19^(Arf) residues 1-64 and human p14^(Arf) residues 1-132. Theability of these peptides to bind Hdm2 fragments was determined usingthe surface plasmon resonance (SPR) technique with a Biacore 3000instrument. His affinity tagged Hdm2 210-304 was immobilized on thesurface of a C-1 chip through capture by a covalently cross-linkedanti-His antibody. Library peptides were allowed to flow over the Hdm2surface, or over a control surface lacking an Hdm2 fragment. Relativebinding affinity was judged on the basis of the maximal SPR signaldetected after binding for 10 minutes. This unusually long inject timewas used because some peptides exhibited slow association kinetics; thelong association time allowed weak binding peptides to be identified.The peptides that exhibited the largest relative binding affinity map tothe N-termini of mouse and human Arf (FIGS. 5a and 5 b); however, thelength of the Hdm2-binding segment for the two is slightly different.For example, the first four peptides from the human library bind Hdm2210-304; these span the segment of human p14^(Arf) that encloses the twoArf motifs. In contrast, 6 of the first 7 peptides of the mousep19^(Arf) library bind Hdm2 210-304. The first five of these enclose thetwo Arf motifs while the last, spanning residues 31-45, lacks elementsof an Arf motif but contains the RRPR motif that is the nucleolarlocalization signal (NoLS) for mouse Arf. One additional human Arfpeptide binds Hdm2 210-304; this spans residues 86-100 and contains theNoLS (with the sequence RRPR) for human Arf. Through an unknownmechanism, the RRPR motif within these nucleolar localization signalscauses the polypeptides to become localized in nucleoli of eukaryoticcells.

[0166] A similar approach using short synthetic peptides and SPR wasused to map the segments of Hdm2 that bind mArfN37 (FIG. 5c). Peptideswithin two segments of the central region of Hdm2 bind with relativelyhigh affinity to mArfN37, including those spanning residues 235-259 and275-289 of SEQ ID NO:8. The first of these as Arf-binding segment istermed “H1” and the second is termed segment “H2”. The peptidecontaining residues 290-304 of SEQ ID NO:8 exhibits modest affinity formArfN37; however, based on results discussed below this appears to bedue to non-specific electrostatic forces.

[0167] To investigate the roles of individual Arf motifs (A1 and A2) andArf-binding segments of Hdm2 (H1 and H2) in the interactions between Arfand Hdm2 the binding of peptides containing these small segments (A1,A2, H1, or H2) to larger protein fragments of the corresponding bindingpartner (mArfN37 or Hdm2 210-304) were monitored using the CD-basedbinding assay. Peptides corresponding to the mouse Arf motifs, A1 (1-14)and A2 (16-30), bind independently to Hdm2 210-304 and induce thetransition from random conformations to β-strand secondary structure.Similarly, peptides containing the human Arf motifs, A1 (1-14) and A2(16-30), bind Hdm2 210-304 and produce the random-to-β-strand structuretransition. Peptides from the Hdm2 library that were positive formArfN37 binding on the basis of SPR (i.e. those within H1 and H2) weremixed with mArfN37 and binding was monitored in the same manner. Threepeptides from the H1 segment and one from the H2 segment of Hdm2 induceβ-strand secondary structure when mixed with mArfN37 (FIG. 2c). Theseexperiments indicate that peptides containing A1, A2, H1 or H2 caninteract with larger fragments of the target protein and that theinteractions also occur through the formation of β-sheet secondarystructure. Peptides lacking A1, A2, H1 or H2 failed to induce thestructural transition. Importantly, however, further reduction in thesize of the interacting species led to a loss of Arf/Hdm2 binding asjudged by a failure to induce structure in CD titration experiments. Forexample, while peptides derived from the A1 or A2 segments of Arf bindHdm2 210-304, they fail to bind short peptides (15 amino acids inlength) derived from the H1 or H2 segments of Hdm2. Similarly, whilepeptides derived from the H1 or H2 segments of Hdm2 bind mArfN37, theyfail to bind peptides derived from the A1 or A2 segments of mouse orhuman Arf. Furthermore, mixtures of two, three, or four differentpeptides that contain the important binding segments (A1, A2, H1, andH2) fail to interact, in contrast to the results obtained when A1 andA2, or H1 and H2, are covalent linked in larger protein fragments.Apparently, cooperative interactions between covalently linked bindingsegments from one protein (A1 and A2 of Arf, or H1 and H2 of Hdm2) areminimally required for Arf/Hdm2 interactions.

[0168] Arf/Hdm2 Interactions Mediate Nucleolar Colocalization.

[0169] Microinjection and live cell imaging were used to determinewhether the Hdm2 domains that were shown to bind Arf in the SPR and CDbinding studies bound to Arf in cells and whether these domains aresequestered within nucleoli by Arf. Hdm2 deletion constructs tagged witha fluorescent label (Texas Red™) were microinjected into the nucleus ofNIH 3T3 cells. importantly, the NIH 3T3 cell line used lacks the genefor Arf. Nuclear microinjection was used because the Hdm2 constructscontaining the central Arf-binding domain lack the nuclear localizationsignal found between residues 181-186. A covalently bound fluorescentlabel was used to detect Hdm2 fragments instead of immuno fluorescenceto avoid nonspecific background staining and staining variability due todifferential antibody reactivity. In addition, preliminary results usingthe antibody of choice to detect Hdm2 210-326 (2A10) [Weber et al., Nat.Cell Biol., 1:20-26 (1999)] gave poor staining results, possibly becausethe antibody epitope is very near the Arf binding site and Arf bindinginhibits antibody binding [Midgley et al., Oncogene, 19:2312-23 (2000)].Arf was delivered to cells prior to microinjection by transfection witha plasmid expressing GFP-p19^(Arf).

[0170] Texas Red™ labeled Hdm2 deletion constructs containing residues210-275, 210-304, 277-350 and 277-491 were individually injected intocells in the absence or presence of the GFP-p19^(Arf) fusion protein. Incells that did not express Arf, all constructs were localized in thenucleoplasm and appeared to be excluded from nucleoli. In contrast, whenGFP-Arf was expressed, three of these constructs that contain all orportions of the central, Arf-binding domain exhibited a distinctnucleolar localization pattern. For example, Hdm2 210-275, 210-304 and277-491 all displayed nucleolar localization when Arf was expressed.Hdm2 277-350 also binds GFP-Arf, but causes the complex to be localizedin the nucleoplasm rather than in nucleoli. These results are consistentwith a report [Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)]showing that Hdm2 constructs that contain a segment spanning residues210-304 bind Arf and are sequestered within nucleoli. The use of livecell imaging also allowed the kinetics of localization to be monitored.Within 5 minutes after injection of Hdm2 constructs, complexrelocalization was complete.

Discussion

[0171] The studies disclosed above focused on understanding themolecular basis of Arf and Hdm2 interactions and their relationship tobiological function. Previously, the domains mediating Arf and Hdm2interactions to the N-terminal 37 amino acids of p19^(Arf) [Weber etal., Nat. Cell Biol., 1:20-26 (1999); Weber et al., Mol. Cell Biol.,20:2517-2528 (2000)] and the central acidic domain of Hdm2 (210-304)[Weber et al., Mol Cell Biol., 20:2517-2528 (2000)] had been localized.Structural analysis of mArfN37 in solution showed that this domain isdynamically disordered in the unbound state [DiGiammarino et al.,Biochem., 40:2379-2386 (2001)]. Interestingly, the Arf interactingdomain of Hdm2 is shown herein to also be dynamically disordered insolution. For example, CD spectra for Hdm2 acidic domain-containingfragments (210-275 and 210-304) show no signs of secondary structure andNMR spectra reveal poorly dispersed resonances consistent with randomcoil chemical shift values. Further, steady-state heteronuclear {¹H}-¹⁵NNOE values for Hdm2 210-275 indicated that amides throughout the entirepolypeptide are highly dynamic. Importantly, as shown herein, thesedisordered Arf and Hdm2 domains bind to each other in both in vitro andcellular assays demonstrating the relevance of the disordered states tobiological function.

[0172] The importance of dynamically disordered proteins or domains inbiological systems, and in the regulation of cell division, is wellestablished [Kriwacki et al., Proc. Natl. Acad. Sci. USA, 93:11504-11509(1996); Uversky et al., Protein Sci., 8:161-73 (1999); Sosnick et al.,Proteins, 24:427-32 (1996); Dyson et al., Biol., 5:499-503 (1998);Plaxco et al., Nature, 386:657-658 (1997); Wright et al., J. Mol Biol,293:321-31 (1999)]. For example, the N-terminal domains of the cyclindependent kinase inhibitors p21 [Kriwacki et al., Proc. Natl. Acad. Sci.USA, 93:11504-11509 (1996); Kriwacki et al., J. Amer. Chem. Soc.,118:5320-5321 (1996)] and p27 are largely unstructured prior to bindingof their cellular targets. Currently, the functional advantage(s) of the‘folding-on-binding’ mechanism is not well understood. Intuitively, theloss of conformational entropy associated with folding will reduce theGibbs free energy of binding for dynamically disordered proteins bindingtheir targets [Kriwacki et al., Proc. Natl. Acad. Sci. USA,93:11504-11509 (1996); Spolar et al., Science, 263:777-84 (1994)]. Ithas been suggested that the advantage of this mechanism is to enhancespecificity [Spolar et al., Science, 263:777-84 (1994)] and/or to allowmultiple, structurally distinct substrates to be bound [Kriwacki et al.,Proc. Natl. Acad. Sci. USA, 93:11504-11509 (1996); Kim et al., Nature,404:151-8 (2000)]. A recent computational study focused on understandingthe impact of conformational entropy in protein folding [Pappu et al.,Proc Natl Acad Sci USA, 97:12565-70 (2000)] suggests that the entropypenalty may not be as great as commonly envisioned due to stericrestrictions by amino acid side chains on the vastness of polypeptideconformational space. While dynamic and highly disordered, flexiblepolypeptides are probably conformationally restrained in solution bysteric and other interaction forces; the challenges for the future areto develop approaches to quantitatively describe these biasedconformations and to relate them to biological function. The need forsuch studies continues to grow as more examples of biologically active,dynamically disordered proteins appear in the literature. Thesignificance of the observations disclosed herein with the Arf:Hdm2system is that, in contrast to previous observations of thefolding-on-binding phenomenon involving a single disordered protein,both components of the Arf:Hdm2 system undergo folding-on-binding.

[0173] β-strands form when dynamically disordered segments of Arf andHdm2 interact. The formation of β-stand secondary structure isaccompanied by the cooperative assembly of large supramolecularstructures. The large size of these assemblies has been confirmed by gelfiltration chromatography and NMR spectroscopy. For example, resonancesfor ¹⁵N-Hdm2 210-304 are broadened beyond detection when an excess ofunlabeled mArfN37 is added to the solution. Arf:Hdm2 complexes appear tobe formed by the cooperative assembly of like structural units intoextended β-networks. This is supported by the appearance of the thermaldenaturation curves for the mArfN37:Hdm2 210-304 assembly, which showthe characteristic sigmoidal shape of a cooperative, two-state proteinunfolding transition [Creighton et al., Proteins: Structures andMolecular Properties, W. H. Freeman & Co., New York, N.Y. (1993)].Thermal unfolding is not reversible, however. Electrostatic forcesstabilize the β-assemblies as shown by the salt-dependence of the T_(m)values.

[0174] Through in vitro binding assays two segments of similar sequencein mouse and human Arf-the consensus for which is termed herein the Arfmotif-that mediate binding to the central acidic domain of Hdm2 andthat, individually, induce the formation of β-strands when mixed withthe central, acidic domain of Hdm2 have been identified herein. Further,peptides derived from the Arf-binding segments of Hdm2-termed H1 andH2-induce β-strands when mixed with mArfN37. Importantly, however, themixing of a short peptide containing either A1 or A2 with anothercontaining either H1 or H2 does not lead to the formation of β-strands.CD was used to monitor the structural effects of peptide mixing and theconcentrations used (1-10 μM) may have been below the threshold forbinding of individual domains. The results disclosed herein with largerprotein fragments containing either contiguous A1-A2 or H1-H2 mixed withshort peptides containing a single binding segment (i.e. H1 or H2, or A1or A2, respectively) indicate that cooperative assembly of β-strandsrequires two binding elements of one protein (Arf or Hdm2) and oneelement of the other. An exception to this is the interaction of Hdm2210-275, which contains only the H1 binding segment, with short peptidescontaining either the human or mouse A1 segment. Hdm2 210-275 maycontain a portion of the H2 segment that participates in cooperativeinteractions with its own H1 segment and the A1 segments of thepeptides.

[0175] Peptides from the human and mouse A1 and A2 segments of Arfinduce β-strand assembly when mixed with Hdm2 210-304. In contrast,peptides from the human and mouse A2 segment of Arf fail to induceβ-strand assembly with Hdm2 210-275 but do induce β-strand assembly withHdm2 210-304. These findings indicate that the H2 binding segmentinteracts only with the A2 segment of Arf. Similarly, H1 may selectivelyinteract with A1 but, based on the data disclosed herein, A1 may alsointeract with H2.

[0176] It is difficult to rank the relative importance of the variousmodes of Arf:Hdm2 interaction (i.e. A1-H1, A2-H2, and A1-H2) with regardto biological function. However, several findings indicate that the A1segment of human and mouse Arf plays a dominant biological role. First,Weber, et al. have examined the effects of deleting the A1 or A2segments of full-length mouse Arf, either individually or incombination, on the ability of mouse Arf to cause cell cycle arrest[Weber et al., Mol. Cell Biol., 20:2517-2528 (2000)]. Deletion of A1, orA1 and A2 together, from mouse Arf almost completely eliminated theability to arrest cell division while deletion of residues 26-37 thatpartially contain the A2 segment produced arrest in some cells but notin others. While it is difficult to quantify the differences in thebiological effects of the different binding site deletion constructs,deletion of the A1 segment uniformly eliminated the ability to arrestcell division and can be ranked as the most essential Hdm2-bindingelement. Second, Midgley, et al., have shown that a GFP fusion proteinlinked to the N-terminal 20 amino acids of human Arf, which contains thehuman A1 segment that is almost identical to that from mouse Arf (FIG.2a), produces Arf-like biological effects in cellular assays [Midgley etal., Oncogene, 19:2312-23 (2000)]. This result has been confirmed byLlanos, et al., using both N- and C-terminal fusions of residues 2-29 ofhuman Arf to GFP. For Hdm2, biological data for constructs withdeletions of the H1 and/or H2 segments is not available. However, theabsolute conservation of amino acids within H1 from humans and mice tozebra fish and tree frogs (FIGS. 2b-2 c) suggests that this segment isimportant for biological function. Amino acids within the H2 segment arealso evolutionarily conserved but not to the same degree as the H1segment. The A1 segment of Arf and H1 of Hdm2 may play dominantbiological roles; these interactions, however, may not be strong enoughto support high affinity interactions and are assisted by interactionsbetween the A2 segment of Arf and H2 of Hdm2 in the formation ofArf:Hdm2 assemblies in cells.

[0177] As further evidence that isolated domains from Arf (containingthe A1 and A2 segments) and Hdm2 (containing the H1 and/or H2 segments)are functionally competent, microinjection and live cell imagingexperiments demonstrate that the domains of Hdm2 that have beencharacterized in vitro herein, retain the ability to interact with Arfin vivo. The Hdm2 constructs 210-304 and 210-275 contain two (H1 and H2)and one (H1 only) of the Hdm2 binding segments, respectively. Afternuclear microinjection, each of these constructs was re-localized to thenucleolus in the presence of GFP-p19^(Arf). Since the Hdm2 NoLS has beendeleted from both of these constructs, the p19^(Arf) NoLS signal must bedriving the nucleolar localization for both proteins. In contrast, theHdm2 construct 277-350 relocalizes Arf to the nucleoplasm. This resultconfirms the presence of an Arf binding motif in Hdm2 beyond amino acid277 and shows that, upon binding of Hdm2 277-350 to GFP-p19A, the ArfNoLS becomes inaccessible to the localization machinery. This result isconsistent with localization experiments performed using full lengthHdm2 [Lohrum et al., Nat. Cell Biol., 2:179-81 (2000); Weber et al.,Mol. Cell Biol., 20:2517-28 (2000)]. Localization of Hdm2 277-491 tonucleoli only in the presence of p19^(Arf) demonstrates that themechanism for exposure of the cryptic Hdm2 NoLS is operative even in theabsence of residues 1-176 of Hdm2. These biological results areconsistent with the existence of two Arf-binding segments within the210-350 segment of Hdm2, as identified through in vitro assays usingshort peptides and protein fragments.

[0178] A novel mechanism of protein-protein interaction mediatesArf:Hdm2 binding. The strict requirement for the presence of bothproteins—Arf and Hdm2—for the formation of β-strand-containingassemblies differentiates this system from others that utilize thefolding-on-binding mechanism. Further, while the Arf:Hdm2 system issimilar to amyloid proteins in that they form extended networkscomprised of β-strands, the individual components of the Arf:Hdm2 systemfail to form β-assemblies alone under a wide range of solutionconditions. While many proteins will form amyloid-like aggregates withP-fibril structure [Booth et al., Nature, 385:787-93 (1997); Ohnishi etal., J. Mol. Biol., 301:477-89 (2000); Alexandrescu and Rathgeb-Szabo,J. Mol Biol, 291:1191-206 (1999); [Esposito et al., Protein Sci.,9:831-845 (2000); Chiti et al., EMBO J, 19:1441-1449 (2000); Wilkins etal., Eur. J. Biochem., 267:2609-2616 (2000)], Arf and Hdm2 formβ-assemblies only when both components are present. Insight into thetwo-component, folding-on-binding phenomenon can be gained byconsidering the unusual amino acid composition of the interactingsegments of Arf and Hdm2 (i.e. A1, and A2, and H1 and H2). As previouslyreported, the N-termini of both human and mouse Arf are unusually richin Arg residues [DiGiammarino et al., Biochem., 40:2379-2386 (2001)].Electrostatic repulsion between these Arg residues may cause thepolypeptide to be dynamically disordered. Interestingly, the Arf-bindingsegments of Hdm2 identified here (H1 and H2) are rich in acidicresidues. The carboxylate groups of these acidic residues probablyinteract with the guanidinium groups of Arg residues within the A1 andA2 segments of Arf via electrostatic interactions. The ability of saltto reduce the T_(m) value for the mArfN37:Hdm2 210-304 assembly stronglysupports the idea that electrostatic interactions stabilize Arf:Hdm2assemblies. In the absence of Arf, the acidic residues within theArf-binding segments of Hdm2 (H1 and H2) repel each other, producing thedynamically disordered conformations observed here. Interestingly, theJpred secondary structure prediction algorithm predicts β-strandconformations exactly within the segments of Arf and Hdm2 which havebeen shown to be important for molecular interactions (FIGS. 2a-2 c).Thus, the sequences within these segments (A1 and A2, and H1 and H2) areconsistent with extended, β-strand conformations. However, the highfrequency of like-charged residues within them may cause the individualpolypeptides to be dynamically disordered. It is also possible that theβ-strand regions of φ, ψ conformational space are populated within theH1 and H2 segments and that the methods of analysis (CD and NMR) usedhave failed to detect them.

[0179] In addition to electrostatic interactions, Arf:Hdm2 interactionsare likely to be mediated by hydrophobic interactions. The Arg residuesof the Arf motif (FIG. 4) are separated by a string of hydrophobicresidues, including a highly conserved segment with the sequence FLV. Inan extended conformation, the arrangement of Arg and hydrophobicresidues within the Arf motif would give rise to both types of residues(hydrophobic and Arg) on both faces of a β-pleated sheet. In the H1segment of Hdm2, acidic residues and hydrophobic residues alternatewithin the sequence (FIG. 2b). This situation would allow acidicresidues of Hdm2 to interact with Arg residues of Arf in the context ofβ-strands composed of intermingled A1 (or A2) and H1 segments.Hydrophobic residues of H1 would align, on the opposite face, withhydrophobic residues of the Arf motif. The conclusion that bothelectrostatic and hydrophobic forces stabilize Arf:Hdm2 β-assemblies isconsistent with their high degree of stability, as exemplified byresistance to denaturation by urea, detergent, salt and extremes of pH.It would appear reasonable that these highly stable structures would befavored within the cellular environment as well as in vitro. It is ofinterest that the nucleolus, where Arf and Hdm2 are localized, wasoriginally characterized by its granular and fibrillar nature [Olson etal., Trends Cell Biol., 10:189-96 (2000)] that is, in principle,consistent with the extended structures reported here for Arf and Hdm2.

[0180] The biological function of Hdm2 is, in part, to maintain p53 atlow levels by actively controlling its ubiquitination, nuclear exportand proteosome-dependent degradation. The early observation that Arfleads to sequestration of Hdm2 within nucleoli suggested that Arfinhibited Hdm2-dependent degradation of p53 by physically separating thedestroyer, Hdm2, from it target, p53. However, whether Arf inhibits thedestroyer function of nucleoplasmic Hdm2, which has access to p53, hasbeen an open question. Llanos, et al., have recently reported thattruncated forms of human Arf that fail to localize within nucleolimaintain the ability to stabilize and activate p53 [Llanos et al., Nat.Cell Biol., 3:445-452 (2001)]. This suggests that the binding of Arf toHdm2 within the nucleoplasm, which may occur prior to nucleolarcolocalization, directly inhibits some or all of the destroyer functionsof Hdm2 toward p53. Hdm2 is comprised of several domains that mediatep53 binding (N-terminal helical domain) [Kussie et al., Science,274:948-953 (1996)], Arf binding (acidic domain studied here), and p53ubiquitination (C-terminal RING domain) [Geyer et al., Nat. Cell Biol.,2:569-73 (2000); Fang et al., J. Biol. Chem., 275:8945-8951 (2000)].Lohrum, et al., have shown that when Arf binds Hdm2, the NoLS of Arf ishidden and a cryptic NoLS within the RING domain of Hdm2 is revealed,leading to nucleolar colocalization [Lohrum et al., Nat. Cell Biol.,2:179-81 (2000)]. Consistent with the RING domain of Hdm2 (and Mdm2)playing an important role in p53 ubiquitination, nuclear export anddegradation [Geyer et al., Nat. Cell Biol., 2:569-73 (2000); Fang etal., J. Biol. Chem., 275:8945-8951 (2000); Honda and Yasuda, Oncogene,19:1473-1476 (2000); Argentini et al., Oncogene, 19:3849-3857 (2000);Boyd et al., Nat. Cell Biol., 2:563-568 (2000)], when Arf binds thecentral, acidic domain of Hdm2 it not only exposes a cryptic NoLS withinthe Hdm2 RING domain but also alters the structure and function of thisdomain in the context of E3 ubiquitin ligase activity. The structure ofthe UbcH7/Cbl E2/E3 complex [Zheng et al., Cell, 102:533-539 (2000)]shows that the RING domain within the Cbl E3 subunit interacts with theUbcH7 E2 and serves to orient the E2 with respect to a small peptidederived from Zap-70, a target of this E2/E3 complex. The RING domain ofHdm2 can play a similar ‘orienting’ role within the E2/E3 complex thattargets p53. The binding of Arf to Hdm2 can alter the conformation ofthe Hdm2 RING domain to expose the cryptic NoLS and inhibit theorienting function of the RING domain. It is likely that intramolecularinteraction between the domains of Hdm2 hide the cryptic NoLS andmaintain the RING domain in an active E3 ubiquitin ligase conformation.Arf binding, through the interactions described herein, can disruptinter-domain interactions, revealing the cryptic NoLS and inhibiting E3ubiquitin ligase activity toward p53.

[0181] While the invention has been described and illustrated herein byreferences to the specific embodiments, various specific material,procedures and examples, it is understood that the invention is notrestricted to the particular material combinations of material, andprocedures selected for that purpose. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0182] It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

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1 25 1 713 DNA Mus musculus CDS (43)..(549) 1 gtcacagtga ggccgccgctgagggagtac agcagcggga gc atg ggt cgc agg 54 Met Gly Arg Arg 1 ttc ttggtc act gtg agg att cag cgc gcg ggc cgc cca ctc caa gag 102 Phe Leu ValThr Val Arg Ile Gln Arg Ala Gly Arg Pro Leu Gln Glu 5 10 15 20 agg gttttc ttg gtg aag ttc gtg cga tcc cgg aga ccc agg aca gcg 150 Arg Val PheLeu Val Lys Phe Val Arg Ser Arg Arg Pro Arg Thr Ala 25 30 35 agc tgc gctctg gct ttc gtg aac atg ttg ttg agg cta gag agg atc 198 Ser Cys Ala LeuAla Phe Val Asn Met Leu Leu Arg Leu Glu Arg Ile 40 45 50 ttg aga aga gggccg cac cgg aat cct gga cca ggt gat gat gat ggg 246 Leu Arg Arg Gly ProHis Arg Asn Pro Gly Pro Gly Asp Asp Asp Gly 55 60 65 caa cgt tca cgt agcagc tct tct gct caa cta cgg tgc aga ttc gaa 294 Gln Arg Ser Arg Ser SerSer Ser Ala Gln Leu Arg Cys Arg Phe Glu 70 75 80 ctg cga gga ccc cac tacctt ctc ccg ccc ggt gca cga cgc agc gcg 342 Leu Arg Gly Pro His Tyr LeuLeu Pro Pro Gly Ala Arg Arg Ser Ala 85 90 95 100 gga agg ctt cct gga cacgct ggt ggt gct gca cgg gtc agg ggc tcg 390 Gly Arg Leu Pro Gly His AlaGly Gly Ala Ala Arg Val Arg Gly Ser 105 110 115 gct gga tgt gcg cga tgcctg ggg tcg cct gcc gct cga ctt ggc cca 438 Ala Gly Cys Ala Arg Cys LeuGly Ser Pro Ala Ala Arg Leu Gly Pro 120 125 130 aga gcg ggg aca tca agacat cgt gcg ata ttt gcg ttc cgc tgg gtg 486 Arg Ala Gly Thr Ser Arg HisArg Ala Ile Phe Ala Phe Arg Trp Val 135 140 145 ctc ttt gtg ttc cgc tgggtg gtc ttt gtg tac cgc tgg gaa cgt cgc 534 Leu Phe Val Phe Arg Trp ValVal Phe Val Tyr Arg Trp Glu Arg Arg 150 155 160 cca gac cga cgg gcatagcttcagc tcaagcacgc ccagggccct ggaacttcgc 589 Pro Asp Arg Arg Ala 165ggccaatccc aagagcagag ctaaatccgg cctcagcccg cctttttctt cttagcttca 649cttctagcga tgctagcgtg tctagcatgt ggctttaaaa aatacataat aatgcttttt 709tttt 713 2 169 PRT Mus musculus 2 Met Gly Arg Arg Phe Leu Val Thr ValArg Ile Gln Arg Ala Gly Arg 1 5 10 15 Pro Leu Gln Glu Arg Val Phe LeuVal Lys Phe Val Arg Ser Arg Arg 20 25 30 Pro Arg Thr Ala Ser Cys Ala LeuAla Phe Val Asn Met Leu Leu Arg 35 40 45 Leu Glu Arg Ile Leu Arg Arg GlyPro His Arg Asn Pro Gly Pro Gly 50 55 60 Asp Asp Asp Gly Gln Arg Ser ArgSer Ser Ser Ser Ala Gln Leu Arg 65 70 75 80 Cys Arg Phe Glu Leu Arg GlyPro His Tyr Leu Leu Pro Pro Gly Ala 85 90 95 Arg Arg Ser Ala Gly Arg LeuPro Gly His Ala Gly Gly Ala Ala Arg 100 105 110 Val Arg Gly Ser Ala GlyCys Ala Arg Cys Leu Gly Ser Pro Ala Ala 115 120 125 Arg Leu Gly Pro ArgAla Gly Thr Ser Arg His Arg Ala Ile Phe Ala 130 135 140 Phe Arg Trp ValLeu Phe Val Phe Arg Trp Val Val Phe Val Tyr Arg 145 150 155 160 Trp GluArg Arg Pro Asp Arg Arg Ala 165 3 540 DNA Homo sapiens CDS (142)..(537)3 cgcgcctgcg gggcggagat gggcaggggg cggtgcgtgg gtcccagtct gcagttaagg 60gggcaggagt ggcgctgctc acctctggtg ccaaagggcg gcgcagcggc tgccgagctc 120ggccctggag gcggcgagaa c atg gtg cgc agg ttc ttg gtg acc ctc cgg 171 MetVal Arg Arg Phe Leu Val Thr Leu Arg 1 5 10 att cgg cgc gcg tgc ggc ccgccg cga gtg agg gtt ttc gtg gtt cac 219 Ile Arg Arg Ala Cys Gly Pro ProArg Val Arg Val Phe Val Val His 15 20 25 atc ccg cgg ctc acg ggg gag tgggca gcg cca ggg gcg ccc gcc gct 267 Ile Pro Arg Leu Thr Gly Glu Trp AlaAla Pro Gly Ala Pro Ala Ala 30 35 40 gtg gcc ctc gtg ctg atg cta ctg aggagc cag cgt cta ggg cag cag 315 Val Ala Leu Val Leu Met Leu Leu Arg SerGln Arg Leu Gly Gln Gln 45 50 55 ccg ctt cct aga aga cca ggt cat gat gatggg cag cgc ccg agt ggc 363 Pro Leu Pro Arg Arg Pro Gly His Asp Asp GlyGln Arg Pro Ser Gly 60 65 70 gga gct gct gct gct cca cgg cgc gga gcc caactg cgc cga ccc cgc 411 Gly Ala Ala Ala Ala Pro Arg Arg Gly Ala Gln LeuArg Arg Pro Arg 75 80 85 90 cac tct cac ccg acc cgt gca cga cgc tgc ccggga ggg ctt cct gga 459 His Ser His Pro Thr Arg Ala Arg Arg Cys Pro GlyGly Leu Pro Gly 95 100 105 cac gct ggt ggt gct gca ccg ggc cgg ggc gcggct gga cgt gcg cga 507 His Ala Gly Gly Ala Ala Pro Gly Arg Gly Ala AlaGly Arg Ala Arg 110 115 120 tgc ctg ggg ccg tct gcc cgt gga cct ggc tga540 Cys Leu Gly Pro Ser Ala Arg Gly Pro Gly 125 130 4 132 PRT Homosapiens 4 Met Val Arg Arg Phe Leu Val Thr Leu Arg Ile Arg Arg Ala CysGly 1 5 10 15 Pro Pro Arg Val Arg Val Phe Val Val His Ile Pro Arg LeuThr Gly 20 25 30 Glu Trp Ala Ala Pro Gly Ala Pro Ala Ala Val Ala Leu ValLeu Met 35 40 45 Leu Leu Arg Ser Gln Arg Leu Gly Gln Gln Pro Leu Pro ArgArg Pro 50 55 60 Gly His Asp Asp Gly Gln Arg Pro Ser Gly Gly Ala Ala AlaAla Pro 65 70 75 80 Arg Arg Gly Ala Gln Leu Arg Arg Pro Arg His Ser HisPro Thr Arg 85 90 95 Ala Arg Arg Cys Pro Gly Gly Leu Pro Gly His Ala GlyGly Ala Ala 100 105 110 Pro Gly Arg Gly Ala Ala Gly Arg Ala Arg Cys LeuGly Pro Ser Ala 115 120 125 Arg Gly Pro Gly 130 5 1710 DNA Mus musculus5 gaggagccgc cgccttctcg tcgctcgagc tctggacgac catggtcgct caggccccgt 60ccgcggggcc tccgcgctcc ccgtgaaggg tcggaagatg cgcgggaagt agcagccgtc 120tgctgggcga gcgggagacc gaccggacac ccctggggga ccctctcgga tcaccgcgct 180tctcctgcgg cctccaggcc aatgtgcaat accaacatgt ctgtgtctac cgagggtgct 240gcaagcacct cacagattcc agcttcggaa caagagactc tggttagacc aaaaccattg 300cttttgaagt tgttaaagtc cgttggagcg caaaacgaca cttacactat gaaagagatt 360atattttata ttggccagta tattatgact aagaggttat atgacgagaa gcagcagcac 420attgtgtatt gttcaaatga tctcctagga gatgtgtttg gagtcccgag tttctctgtg 480aaggagcaca ggaaaatata tgcaatgatc tacagaaatt tagtggctgt aagtcagcaa 540gactctggca catcgctgag tgagagcaga cgtcagcctg aaggtgggag tgatctgaag 600gatcctttgc aagcgccacc agaagagaaa ccttcatctt ctgatttaat ttctagactg 660tctacctcat ctagaaggag atccattagt gagacagaag agaacacaga tgagctacct 720ggggagcggc accggaagcg ccgcaggtcc ctgtcctttg atccgagcct gggtctgtgt 780gagctgaggg agatgtgcag cggcggcacg agcagcagta gcagcagcag cagcgagtcc 840acagagacgc cctcgcatca ggatcttgac gatggcgtaa gtgagcattc tggtgattgc 900ctggatcagg attcagtttc tgatcagttt agcgtggaat ttgaagttga gtctctggac 960tcggaagatt acagcctgag tgacgaaggg cacgagctct cagatgagga tgatgaggtc 1020tatcgggtca cagtctatca gacaggagaa agcgatacag actcttttga aggagatcct 1080gagatttcct tagctgacta ttggaagtgt acctcatgca atgaaatgaa tcctcccctt 1140ccatcacact gcaaaagatg ctggaccctt cgtgagaact ggcttccaga cgataagggg 1200aaagataaag tggaaatctc tgaaaaagcc aaactggaaa actcagctca ggcagaagaa 1260ggcttggatg tgcctgatgg caaaaagctg acagagaatg atgctaaaga gccatgtgct 1320gaggaggaca gcgaggagaa ggccgaacag acgcccctgt cccaggagag tgacgactat 1380tcccaaccat cgacttccag cagcattgtt tatagcagcc aagaaagcgt gaaagagttg 1440aaggaggaaa cgcagcacaa agacgagagt gtggaatcta gcttctccct gaatgccatc 1500gaaccatgtg tgatctgcca ggggcggcct aaaaatggct gcattgttca cggcaagact 1560ggacacctca tgtcatgttt cacgtgtgca aagaagctaa aaaaaagaaa caagccctgc 1620ccagtgtgca gacagccaat ccaaatgatt gtgctaagtt acttcaacta gctgacctgc 1680tcacaaaaat agaattttat atttctaact 1710 6 489 PRT Mus musculus 6 Met CysAsn Thr Asn Met Ser Val Ser Thr Glu Gly Ala Ala Ser Thr 1 5 10 15 SerGln Ile Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro 20 25 30 LeuLeu Leu Lys Leu Leu Lys Ser Val Gly Ala Gln Asn Asp Thr Tyr 35 40 45 ThrMet Lys Glu Ile Ile Phe Tyr Ile Gly Gln Tyr Ile Met Thr Lys 50 55 60 ArgLeu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp 65 70 75 80Leu Leu Gly Asp Val Phe Gly Val Pro Ser Phe Ser Val Lys Glu His 85 90 95Arg Lys Ile Tyr Ala Met Ile Tyr Arg Asn Leu Val Ala Val Ser Gln 100 105110 Gln Asp Ser Gly Thr Ser Leu Ser Glu Ser Arg Arg Gln Pro Glu Gly 115120 125 Gly Ser Asp Leu Lys Asp Pro Leu Gln Ala Pro Pro Glu Glu Lys Pro130 135 140 Ser Ser Ser Asp Leu Ile Ser Arg Leu Ser Thr Ser Ser Arg ArgArg 145 150 155 160 Ser Ile Ser Glu Thr Glu Glu Asn Thr Asp Glu Leu ProGly Glu Arg 165 170 175 His Arg Lys Arg Arg Arg Ser Leu Ser Phe Asp ProSer Leu Gly Leu 180 185 190 Cys Glu Leu Arg Glu Met Cys Ser Gly Gly ThrSer Ser Ser Ser Ser 195 200 205 Ser Ser Ser Glu Ser Thr Glu Thr Pro SerHis Gln Asp Leu Asp Asp 210 215 220 Gly Val Ser Glu His Ser Gly Asp CysLeu Asp Gln Asp Ser Val Ser 225 230 235 240 Asp Gln Phe Ser Val Glu PheGlu Val Glu Ser Leu Asp Ser Glu Asp 245 250 255 Tyr Ser Leu Ser Asp GluGly His Glu Leu Ser Asp Glu Asp Asp Glu 260 265 270 Val Tyr Arg Val ThrVal Tyr Gln Thr Gly Glu Ser Asp Thr Asp Ser 275 280 285 Phe Glu Gly AspPro Glu Ile Ser Leu Ala Asp Tyr Trp Lys Cys Thr 290 295 300 Ser Cys AsnGlu Met Asn Pro Pro Leu Pro Ser His Cys Lys Arg Cys 305 310 315 320 TrpThr Leu Arg Glu Asn Trp Leu Pro Asp Asp Lys Gly Lys Asp Lys 325 330 335Val Glu Ile Ser Glu Lys Ala Lys Leu Glu Asn Ser Ala Gln Ala Glu 340 345350 Glu Gly Leu Asp Val Pro Asp Gly Lys Lys Leu Thr Glu Asn Asp Ala 355360 365 Lys Glu Pro Cys Ala Glu Glu Asp Ser Glu Glu Lys Ala Glu Gln Thr370 375 380 Pro Leu Ser Gln Glu Ser Asp Asp Tyr Ser Gln Pro Ser Thr SerSer 385 390 395 400 Ser Ile Val Tyr Ser Ser Gln Glu Ser Val Lys Glu LeuLys Glu Glu 405 410 415 Thr Gln His Lys Asp Glu Ser Val Glu Ser Ser PheSer Leu Asn Ala 420 425 430 Ile Glu Pro Cys Val Ile Cys Gln Gly Arg ProLys Asn Gly Cys Ile 435 440 445 Val His Gly Lys Thr Gly His Leu Met SerCys Phe Thr Cys Ala Lys 450 455 460 Lys Leu Lys Lys Arg Asn Lys Pro CysPro Val Cys Arg Gln Pro Ile 465 470 475 480 Gln Met Ile Val Leu Ser TyrPhe Asn 485 7 2372 DNA Homo sapiens 7 gcaccgcgcg agcttggctg cttctggggcctgtgtggcc ctgtgtgtcg gaaagatgga 60 gcaagaagcc gagcccgagg ggcggccgcgacccctctga ccgagatcct gctgctttcg 120 cagccaggag caccgtccct ccccggattagtgcgtacga gcgcccagtg ccctggcccg 180 gagagtggaa tgatccccga ggcccagggcgtcgtgcttc cgcagtagtc agtccccgtg 240 aaggaaactg gggagtcttg agggacccccgactccaagc gcgaaaaccc cggatggtga 300 ggagcaggca aatgtgcaat accaacatgtctgtacctac tgatggtgct gtaaccacct 360 cacagattcc agcttcggaa caagagaccctggttagacc aaagccattg cttttgaagt 420 tattaaagtc tgttggtgca caaaaagacacttatactat gaaagaggtt cttttttatc 480 ttggccagta tattatgact aaacgattatatgatgagaa gcaacaacat attgtatatt 540 gttcaaatga tcttctagga gatttgtttggcgtgccaag cttctctgtg aaagagcaca 600 ggaaaatata taccatgatc tacaggaacttggtagtagt caatcagcag gaatcatcgg 660 actcaggtac atctgtgagt gagaacaggtgtcaccttga aggtgggagt gatcaaaagg 720 accttgtaca agagcttcag gaagagaaaccttcatcttc acatttggtt tctagaccat 780 ctacctcatc tagaaggaga gcaattagtgagacagaaga aaattcagat gaattatctg 840 gtgaacgaca aagaaaacgc cacaaatctgatagtatttc cctttccttt gatgaaagcc 900 tggctctgtg tgtaataagg gagatatgttgtgaaagaag cagtagcagt gaatctacag 960 ggacgccatc gaatccggat cttgatgctggtgtaagtga acattcaggt gattggttgg 1020 atcaggattc agtttcagat cagtttagtgtagaatttga agttgaatct ctcgactcag 1080 aagattatag ccttagtgaa gaaggacaagaactctcaga tgaagatgat gaggtatatc 1140 aagttactgt gtatcaggca ggggagagtgatacagattc atttgaagaa gatcctgaaa 1200 tttccttagc tgactattgg aaatgcacttcatgcaatga aatgaatccc ccccttccat 1260 cacattgcaa cagatgttgg gcccttcgtgagaattggct tcctgaagat aaagggaaag 1320 ataaagggga aatctctgag aaagccaaactggaaaactc aacacaagct gaagagggct 1380 ttgatgttcc tgattgtaaa aaaactatagtgaatgattc cagagagtca tgtgttgagg 1440 aaaatgatga taaaattaca caagcttcacaatcacaaga aagtgaagac tattctcagc 1500 catcaacttc tagtagcatt atttatagcagccaagaaga tgtgaaagag tttgaaaggg 1560 aagaaaccca agacaaagaa gagagtgtggaatctagttt gccccttaat gccattgaac 1620 cttgtgtgat ttgtcaaggt cgacctaaaaatggttgcat tgtccatggc aaaacaggac 1680 atcttatggc ctgctttaca tgtgcaaagaagctaaagaa aaggaataag ccctgcccag 1740 tatgtagaca accaattcaa atgattgtgctaacttattt cccctagttg acctgtctat 1800 aagagaatta tatatttcta actatataaccctaggaatt tagacaacct gaaatttatt 1860 cacatatatc aaagtgagaa aatgcctcaattcacataga tttcttctct ttagtataat 1920 tgacctactt tggtagtgga atagtgaatacttactataa tttgacttga atatgtagct 1980 catcctttac accaactcct aattttaaataatttctact ctgtcttaaa tgagaagtac 2040 ttggtttttt ttttcttaaa tatgtatatgacatttaaat gtaacttatt attttttttg 2100 agaccgagtc ttgctctgtt acccaggctggagtgcagtg ggtgatcttg gctcactgca 2160 agctctgccc tccccgggtt cgcaccattctcctgcctca gcctcccaat tagcttggcc 2220 tacagtcatc tgccaccaca cctggctaattttttgtact tttagtagag acagggtttc 2280 accgtgttag ccaggatggt ctcgatctcctgacctcgtg atccgcccac ctcggcctcc 2340 caaagtgctg ggattacagg catgagccaccg 2372 8 491 PRT Homo sapiens 8 Met Cys Asn Thr Asn Met Ser Val Pro ThrAsp Gly Ala Val Thr Thr 1 5 10 15 Ser Gln Ile Pro Ala Ser Glu Gln GluThr Leu Val Arg Pro Lys Pro 20 25 30 Leu Leu Leu Lys Leu Leu Lys Ser ValGly Ala Gln Lys Asp Thr Tyr 35 40 45 Thr Met Lys Glu Val Leu Phe Tyr LeuGly Gln Tyr Ile Met Thr Lys 50 55 60 Arg Leu Tyr Asp Glu Lys Gln Gln HisIle Val Tyr Cys Ser Asn Asp 65 70 75 80 Leu Leu Gly Asp Leu Phe Gly ValPro Ser Phe Ser Val Lys Glu His 85 90 95 Arg Lys Ile Tyr Thr Met Ile TyrArg Asn Leu Val Val Val Asn Gln 100 105 110 Gln Glu Ser Ser Asp Ser GlyThr Ser Val Ser Glu Asn Arg Cys His 115 120 125 Leu Glu Gly Gly Ser AspGln Lys Asp Leu Val Gln Glu Leu Gln Glu 130 135 140 Glu Lys Pro Ser SerSer His Leu Val Ser Arg Pro Ser Thr Ser Ser 145 150 155 160 Arg Arg ArgAla Ile Ser Glu Thr Glu Glu Asn Ser Asp Glu Leu Ser 165 170 175 Gly GluArg Gln Arg Lys Arg His Lys Ser Asp Ser Ile Ser Leu Ser 180 185 190 PheAsp Glu Ser Leu Ala Leu Cys Val Ile Arg Glu Ile Cys Cys Glu 195 200 205Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu 210 215220 Asp Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser 225230 235 240 Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu AspSer 245 250 255 Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser AspGlu Asp 260 265 270 Asp Glu Val Tyr Gln Val Thr Val Tyr Gln Ala Gly GluSer Asp Thr 275 280 285 Asp Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu AlaAsp Tyr Trp Lys 290 295 300 Cys Thr Ser Cys Asn Glu Met Asn Pro Pro LeuPro Ser His Cys Asn 305 310 315 320 Arg Cys Trp Ala Leu Arg Glu Asn TrpLeu Pro Glu Asp Lys Gly Lys 325 330 335 Asp Lys Gly Glu Ile Ser Glu LysAla Lys Leu Glu Asn Ser Thr Gln 340 345 350 Ala Glu Glu Gly Phe Asp ValPro Asp Cys Lys Lys Thr Ile Val Asn 355 360 365 Asp Ser Arg Glu Ser CysVal Glu Glu Asn Asp Asp Lys Ile Thr Gln 370 375 380 Ala Ser Gln Ser GlnGlu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser 385 390 395 400 Ser Ser IleIle Tyr Ser Ser Gln Glu Asp Val Lys Glu Phe Glu Arg 405 410 415 Glu GluThr Gln Asp Lys Glu Glu Ser Val Glu Ser Ser Leu Pro Leu 420 425 430 AsnAla Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro Lys Asn Gly 435 440 445Cys Ile Val His Gly Lys Thr Gly His Leu Met Ala Cys Phe Thr Cys 450 455460 Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro Cys Pro Val Cys Arg Gln 465470 475 480 Pro Ile Gln Met Ile Val Leu Thr Tyr Phe Pro 485 490 9 8 PRTMus musculus 9 Arg Arg Phe Leu Val Thr Val Arg 1 5 10 9 PRT Mus musculus10 Arg Val Phe Leu Val Lys Phe Val Arg 1 5 11 8 PRT Homo sapiens 11 ArgArg Phe Leu Val Thr Leu Arg 1 5 12 9 PRT Homo sapiens 12 Arg Val Phe ValVal His Ile Pro Arg 1 5 13 9 PRT CONSENSUS SEQUENCE MISC_FEATURE(2)..(2) X can be R or V 13 Arg Xaa Phe Xaa Val Xaa Xaa Xaa Arg 1 5 14 4PRT Nucleolar Localization Sequence 14 Arg Arg Pro Arg 1 15 37 PRT Homosapiens 15 Met Val Arg Arg Phe Leu Val Thr Leu Arg Ile Arg Arg Ala CysGly 1 5 10 15 Pro Pro Arg Val Arg Val Phe Val Val His Ile Pro Arg LeuThr Gly 20 25 30 Glu Trp Ala Ala Pro 35 16 37 PRT Mus musculus 16 MetGly Arg Arg Phe Leu Val Thr Val Arg Ile Gln Arg Ala Gly Arg 1 5 10 15Pro Leu Gln Glu Arg Val Phe Leu Val Lys Phe Val Arg Ser Arg Arg 20 25 30Pro Arg Thr Ala Ser 35 17 37 PRT opossum 17 Met Ile Arg Arg Val Arg ValThr Val Arg Val Ser Arg Ala Cys Arg 1 5 10 15 Pro His His Val Arg IlePhe Val Ala Lys Ile Val Gln Ala Leu Cys 20 25 30 Arg Ala Ser Ala Ser 3518 95 PRT Homo sapiens 18 Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro SerAsn Pro Asp Leu Asp 1 5 10 15 Ala Gly Val Ser Glu His Ser Gly Asp TrpLeu Asp Gln Asp Ser Val 20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu ValGlu Ser Leu Asp Ser Glu 35 40 45 Asp Tyr Ser Leu Ser Glu Glu Gly Gln GluLeu Ser Asp Glu Asp Asp 50 55 60 Glu Val Tyr Gln Val Thr Val Tyr Gln AlaGly Glu Ser Asp Thr Asp 65 70 75 80 Ser Phe Glu Glu Asp Pro Glu Ile SerLeu Ala Asp Tyr Trp Lys 85 90 95 19 95 PRT Mus musculus 19 Ser Ser SerSer Glu Ser Thr Glu Thr Pro Ser His Gln Asp Leu Asp 1 5 10 15 Asp GlyVal Ser Glu His Ser Gly Asp Cys Leu Asp Gln Asp Ser Val 20 25 30 Ser AspGln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser Glu 35 40 45 Asp TyrSer Leu Ser Asp Glu Gly His Glu Leu Ser Asp Glu Asp Asp 50 55 60 Glu ValTyr Arg Val Thr Val Tyr Gln Thr Gly Glu Ser Asp Thr Asp 65 70 75 80 SerPhe Glu Gly Asp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys 85 90 95 20 95PRT hamster 20 Ser Ser Ser Ser Glu Ser Thr Asp Thr Pro Ser Asn Gln AspLeu Asp 1 5 10 15 Asp Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp GlnAsp Ser Val 20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser LeuAsp Ser Glu 35 40 45 Asp Tyr Ser Leu Ser Glu Gly Gly Gln Glu Leu Ser AspGlu Asp Asp 50 55 60 Glu Val Tyr Arg Val Thr Val Tyr Gln Ser Gly Glu SerAsp Val Asp 65 70 75 80 Ser Phe Glu Gly Asp Pro Glu Ile Ser Leu Ala AspTyr Trp Lys 85 90 95 21 95 PRT horse 21 Ser Ser Ser Ser Glu Ser Thr GlyThr Pro Ser Asn Pro Asp Leu Asp 1 5 10 15 Ala Gly Val Ser Glu His SerGly Asp Trp Leu Asp Gln Asp Ser Val 20 25 30 Ser Asp Gln Phe Ser Val GluPhe Glu Val Glu Ser Leu Asp Ser Glu 35 40 45 Asp Tyr Ser Leu Ser Glu GluGly Gln Glu Leu Ser Asp Glu Asp Asp 50 55 60 Glu Val Tyr Arg Val Thr ValTyr Gln Ala Gly Glu Ser Asp Thr Asp 65 70 75 80 Ser Phe Glu Glu Asp ProGlu Ile Ser Leu Ala Asp Tyr Trp Lys 85 90 95 22 95 PRT dog 22 Ser SerSer Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu Asp 1 5 10 15 AlaGly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser Val 20 25 30 SerAsp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser Glu 35 40 45 AspTyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu Asp Asp 50 55 60 GluVal Tyr Arg Val Thr Val Tyr Gln Ala Gly Glu Ser Asp Thr Asp 65 70 75 80Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys 85 90 95 2395 PRT chicken 23 Ser Asn Ser Ser Asp Ser Thr Asp Ser Val Ser Ile ProAsp Leu Asp 1 5 10 15 Ala Ser Ser Leu Ser Glu Asn Ser Asp Trp Phe AspHis Gly Ser Val 20 25 30 Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu SerIle Tyr Ser Glu 35 40 45 Asp Tyr Ser His Asn Glu Glu Gly Gln Glu Leu ThrAsp Glu Asp Asp 50 55 60 Glu Val Tyr Gln Leu Thr Ile Tyr Gln Asp Glu AspSer Asp Ser Asp 65 70 75 80 Ser Phe Asn Glu Asp Pro Glu Ile Ser Leu AlaAsp Tyr Trp Lys 85 90 95 24 90 PRT zebrafish 24 Arg Gly Asn Ser Glu SerSer Asp Ala Asn Ser Asn Ser Asp Val Gly 1 5 10 15 Ile Ser Arg Ser GluGly Ser Glu Glu Ser Glu Asp Ser Asp Ser Asp 20 25 30 Ser Asp Asn Phe SerVal Glu Phe Glu Val Glu Ser Ile Asn Ser Asp 35 40 45 Ala Tyr Ser Glu AsnAsp Val Asp Ser Val Pro Gly Glu Asn Glu Ile 50 55 60 Tyr Glu Val Thr IlePhe Ala Glu Asp Glu Asp Ser Phe Asp Glu Asp 65 70 75 80 Thr Glu Ile ThrGlu Ala Asp Tyr Lys Trp 85 90 25 98 PRT treefrog 25 Gly Leu Arg Cys AspArg Asn Ser Ser Glu Ser Thr Asp Ser Ser Ser 1 5 10 15 Asn Ser Asp ProGlu Arg His Ser Thr Asn Asp Asn Ser Glu His Asp 20 25 30 Ser Asp Gln PheSer Val Glu Phe Glu Val Glu Ser Val Cys Ser Asp 35 40 45 Asp Tyr Ser ProSer Gly Asp Glu His Gly Val Ser Glu Glu Glu Glu 50 55 60 Ile Asn Asp GluVal Tyr Gln Val Thr Ile Tyr Glu Thr Glu Glu Ser 65 70 75 80 Glu Thr AspSer Phe Asp Val Asp Thr Glu Ile Ser Glu Ala Asp Tyr 85 90 95 Trp Lys

What is claimed is:
 1. A method of identifying a compound that caninduce the formation of β-strand assembly of Dm2 comprising: (a)contacting the compound with Dm2 or an inducible fragment of Dm2; and(b) determining whether Dm2 or the inducible fragment of Dm2 is inducedto form a β-strand assembly; wherein a compound is identified when Dm2or the inducible fragment of Dm2 is induced to form a β-strand assembly.2. The method of claim 1 wherein said determining is performed bycircular dichroism measurements.
 3. The method of claim 1 wherein saiddetermining is performed by nuclear magnetic resonance.
 4. The method ofclaim 1 wherein said determining is performed by Fourier TransformInfra-red spectroscopy.
 5. The method of claim 1 wherein saiddetermining is performed by fluorescence spectroscopy.
 6. The method ofclaim 5, wherein said determining is performed by monitoring thefluorescence of a native tryptophan of Dm2 or of the inducible fragmentof Dm2.
 7. The method of claim 5, wherein Dm2 or the inducible fragmentof Dm2 is labeled with a fluorescent probe, and wherein said determiningis performed by monitoring the fluorescence of the fluorescent probe. 8.The method of claim 1 wherein the Dm2 is Hdm2 having the amino acidsequence of SEQ ID NO:8.
 9. The method of claim 1 wherein the induciblefragment of Dm2 comprises amino acid residues 235-259 of SEQ ID NO:8,the H1 segment.
 10. The method of claim 9 wherein the inducible fragmentof Dm2 further comprises amino acid residues 275-289 of SEQ ID NO:8, theH2 segment.
 11. The method of claim 1 wherein the inducible fragment ofDm2 comprises amino acid residues 275-289 of SEQ ID NO:8, the H2segment.
 12. A compound identified by the method of claim 1; whereinsaid compound is not a peptide comprising five or more consecutive aminoacids comprised by a naturally occurring protein.
 13. A method ofidentifying a compound that can enhance the rate of β-strand assembly ofDm2 induced by Arf comprising: (a) contacting the compound with Dm2 oran inducible fragment of Dm2, and Arf or an inducing fragment of Arf;and (b) determining the rate of the β-strand assembly of Dm2 or of theinducible fragment of Dm2; wherein a compound is identified when therate of the β-strand assembly of Dm2 or of the inducible fragment of Dm2increases in the presence of the compound relative to in the absence ofthe compound.
 14. A method of identifying a compound that can inhibitthe formation of β-strand assembly of Dm2 comprising: (a) contacting thecompound with Dm2 or an inducible fragment of Dm2, and Arf or aninducing fragment of Arf; and (b) determining the rate of formation of aβ-strand assembly of Dm2 or the inducible fragment of Dm2; wherein whenthe rate of formation of the β-strand assembly of Dm2 or the induciblefragment of Dm2 decreases in the presence of the compound relative to inits absence, the compound is identified as a compound that can inhibitthe formation of β-strand assembly of Dm2.
 15. A method of identifying acompound that can inhibit the formation of β-strand assembly of Dm2comprising: (a) contacting the compound with Dm2 or an induciblefragment of Dm2, and Arf or an inducing fragment of Arf; and (b)determining the amount of formation of a β-strand assembly of Dm2 or theinducible fragment of Dm2; wherein when the amount of formation of theβ-strand assembly of Dm2 or the inducible fragment of Dm2 decreases inthe presence of the compound relative to in its absence, the compound isidentified as a compound that can inhibit the formation of β-strandassembly of Dm2.
 16. A method of identifying a compound that can inducethe formation of supramolecular assemblies comprised of β-strands of Dm2comprising: (a) contacting the compound with Dm2 or an induciblefragment of Dm2; and (b) determining whether Dm2 or the induciblefragment of Dm2 is induced to form supramolecular assemblies comprisedof β-strands of Dm2; wherein when Dm2 or the inducible fragment of Dm2is induced to form supramolecular assemblies the compound is identifiedas a compound that can induce the formation of supramolecular assembliescomprised of β-strands of Dm2
 17. The method of claim 16 wherein saiddetermining is performed by size exclusion determinations.
 18. Themethod of claim 16 wherein the Dm2 is Hdm2 having the amino acidsequence of SEQ ID NO:8.
 19. The method of claim 16 wherein the fragmentof Dm2 comprises amino acid residues 235-259 of SEQ ID NO:8, the H1segment.
 20. The method of claim 19 wherein the fragment of Dm2 furthercomprises amino acid residues 275-289 of SEQ ID NO:8, the H2 segment.21. The method of claim 16 wherein the fragment of Dm2 comprises aminoacid residues 275-289 of SEQ ID NO:8, the H2 segment.
 22. A compoundidentified by the method of claim 16; wherein said compound is not apeptide comprising five or more consecutive amino acids comprised by anaturally occurring protein.
 23. A method of treating a patient withcancer or a predisposition for getting cancer comprising administeringto the patient a compound that can induce β-strand assembly of Hdm2 in acell.
 24. The method of claim 23 wherein the patient has a tumor withcells that are characterized by a lack of sufficient Arf activity toinduce cell cycle arrest and/or apoptosis; but wherein the cells retainfunctional p53.
 25. A kit for identifying a compound that can induceβ-strand assembly of Dm2 comprising: (a) a peptide comprising an aminoacid sequence selected from the group consisting of amino acid residues235-259 of SEQ ID NO:8; amino acid residues 275-289 of SEQ ID NO:8, andboth amino acid residues 235-259 and amino acid residues 275-289 of SEQID NO:8; and (b) a peptide that comprises two copies of the Arf motifcomprising the amino acid sequence of SEQ ID NO:13.
 26. The kit of claim25 further comprising instructions for identifying a compound that caninduce β-strand assembly of Dm2.
 27. An antibody raised against apeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:13, amino acid residues 235-259 of SEQ ID NO:8,and amino acid residues 275-289 of SEQ ID NO:8.
 28. The antibody ofclaim 26 that is a humanized antibody.
 29. A method of inducingapoptosis in a cell by administering the antibody of claim 28 to thecell.
 30. A method of treating a patient having a tumor comprisingadministering the antibody of claim 29 to the patient; wherein the tumorcontains cells characterized by having functional Arf, functional Hdm2and functional p53.
 31. A method of designing a compound that ispredicted to mimic the ability of Arf to induce the formation of theβ-strand assembly of Dm2, said method comprising: (a) generating acomputer model of a structure of an Arf-Dm2 complex based on: (i) theamino acid sequence of the portions of Arf and Dm2 involved in theArf-Dm2 complex; and (ii) the circular dichroism and Fourier TransformInfra-red spectra obtained for the Arf-Dm2 complex; and (b) designing acompound to bind to Dm2 as Arf does using the computer model of thestructure of the Arf-Dm2 binding complex generated in step (a); whereinsaid compound is predicted to mimic the ability of Arf to induce theformation of the β-strand assembly of Dm2.
 32. The method of claim 31,further comprising: (c) organically synthesizing said compound; (d)contacting the synthesized compound with a Dm2 or an inducible fragmentof Dm2; and (e) determining whether the Dm2 or the inducible fragment ofDm2 has formed of a β-strand assembly; wherein when the Dm2 or theinducible fragment of Dm2 is induced to form a β-strand assembly in step(d), the synthesized compound is identified as a compound that mimicsthe ability of Arf to induce the formation of the β-strand assembly ofDm2.
 33. A peptide consisting of the amino acid sequence of SEQ IDNO:13.
 34. The peptide of claim 33 consisting of the amino acid sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11 and SEQ ID NO:12.
 35. A fusion protein comprising a peptideconsisting of the amino acid sequence of SEQ ID NO:13.
 36. A peptideconsisting of two segments of an Arf protein, wherein each segmentconsists of the amino acid sequence of SEQ ID NO:13.
 37. The peptide ofclaim 36 wherein at least one segment consists of the amino acidsequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11 and SEQ ID NO:12.
 38. The peptide of claim 37wherein the other segment consists of the amino acid sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 andSEQ ID NO:12.
 39. A fusion protein comprising a peptide consisting oftwo segments of an Arf protein, wherein each segment consists of theamino acid sequence of SEQ ID NO:13.
 40. A peptide consisting of aminoacid residues 235-259 of SEQ ID NO:8, the H1 segment.
 41. A fusionprotein comprising a peptide consisting of amino acid residues 235-259of SEQ ID NO:8.
 42. A peptide consisting of amino acid residues 275-289of SEQ ID NO:8.
 43. A fusion protein comprising a peptide consisting ofamino acid residues 275-289 of SEQ ID NO:8.
 44. A peptide consisting ofamino acid residues 235-259 and amino acid residues 275-289 of SEQ IDNO:8.
 45. A fusion protein comprising a peptide consisting of amino acidresidues 235-259 and amino acid residues 275-289 of SEQ ID NO:8.
 46. Acomposition comprising two segments of an Arf protein chemically joinedvia a non-peptide linkage, wherein each segment comprises the amino acidsequence of SEQ ID NO:13.